Compositions and methods for allergen detection

ABSTRACT

The present invention is drawn to nucleic acid aptamer based signaling polynucleotides (SPNs) for allergen detection in samples. Disclosed herein include compositions, compounds, assays and methods of using said SPNs to detect one or more allergens in a sample, particularly food allergens in a food product.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 U.S. National Stage Entry ofInternational Application No. PCT/US2016/029356 filed on Apr. 26, 2016,which claims priority to U.S. Provisional application No. 62/154,200filed on Apr. 29, 2015, the contents of which are each incorporatedherein by reference in their entirety.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSEQLST20661002US371.txt, created on Oct. 24, 2017, which is 10,577 bytesin size. The information in the electronic format of the sequencelisting is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to aptamer based signaling polynucleotides(SPNs), compositions comprising such SPNs, assays and methods of usingsuch SPNs for detection of a protein target, in particular a foodallergen.

BACKGROUND OF THE INVENTION

Allergy is a serious medical condition affecting millions of peopleworldwide, with about 15 million people in the United States, includingmany children. During an allergic reaction, the immune system mistakenlytargets an allergen as a threat and attacks it. The allergic reactionmay affect the skin, the digestive system, the gastrointestinal tract,the respiratory system, the circulatory system and the cardiovascularsystem; and in some allergic reactions, multiple organ systems areaffected. Allergic reactions range from mild to severe orlife-threatening. Severe symptoms may include difficulty in breathing,low blood pressure, chest pain, loss of consciousness, and anaphylaxis.Food allergies are a major health issue in all industrialized countries.People having food allergies currently manage their allergies byavoiding any food that might contain that specific allergen. Theserestrictions have a major impact on the patients' quality of life andthere remains no method for assessing the true allergen content of food.In the United States, food allergy symptoms send someone to theemergency room every three minutes.

Allergen detection is important for many reasons. A fast and accuratedetection method and a portable device that can be easily operated by aperson with food allergies to test their food and determine accuratelyand immediately the allergen content will be beneficial to provide foran informed decision on whether to consume or not. In food industry,allergen detection is critical to ensure accuracy of food labeling andto clean contaminants effectively during food production.

Currently available methods for detecting allergens mostly useantibodies based immunochemical methods (e.g., ELISA, lateral flowdevices), peptides (e.g., mass spectrometry), enzymes, DNA based methods(e.g., PCR) and other generic/non-specific methods (e.g., visualinspection, ATP tests). These methodologies sometime are very complex,expensive, time consuming and unreliable. A fast and accurate method fordetermining the absence/presence of an allergen would be of greatbenefit. Ultrasensitive detection molecules that can detect a trace ofan allergen(s) would be essential for developing a sensitive detectionmethod.

Aptamers, which are single stranded (ss) DNA and RNA molecules, can bindto their targets due to their specific three dimensional structures;they offer specific properties which favor them as new detectionmolecules for protein recognition including allergens. Aptamers andaptamer-based assays have been shown, among many other usefulapplications (e.g., diagnostic tests) as a promising alternative in foodsafety control. A recent review describes analytical strategiesdeveloped using aptamers for the control of pathogens, allergens,adulterants, toxins and other forbidden contaminants to ensure foodsafety (Amaya-Gonzalez, et al., Aptamer-Based Analysis: A PromisingAlternative for Food Safety Control, Sensors, 2013, 13:16292-16311; andAmaya-Gonzalez, et al., Aptamer binding to coelic disease-triggeringhydrophobic proteins: Towards a sensitive gluten detection system. Anal.Chem. 2014, 86(5), 2733-2739). A method of detection of gluten is alsodescribed in PCT Publication PCT/ES2013/000133, 28 Jun. 2013, toAmaya-Gonzalez, et al. Other examples include PCT applicationpublication NOs.: WO2013064818 and WO2012081908 (aptamers thatspecifically bind Staphylococcus aureus); WO2012081906 (aptamers forompc protein in salmonella tiphimirium strain); WO2009070749 (aptamersfor detecting salmonella contamination); and U.S. Pat. Nos. 7,645,582and 7,838,242 (aptamers that bind to listeria surface proteins) (each ofwhich is incorporated herein by reference in its entirety.)

The present invention provides new aptamer based signalingpolynucleotides, compositions comprising such SPNs, and fast, sensitiveand accurate assays to detect the absence or presence of allergens,and/or to quantitatively measure the amount of allergen in test samples.The signaling polynucleotides and detection assays developed in thepresent disclosure may be used in any allergen detection devices in theart, such as microfluidic chips taught in U.S. Pat. No. 8,617,903 andportable devices taught in the commonly owned PCT patent applicationNO.: PCT/US14/62656 filed on Oct. 28, 2014, and U.S. provisionalapplication No. 62/133,632 filed on Mar. 16, 2015 (each of which isincorporated herein by reference in its entirety).

SUMMARY OF THE INVENTION

The present invention relates to compositions, compounds, assays andmethods for detecting one or more allergens in a sample. In someembodiments, allergens are food allergens.

In some embodiments, compositions of the present invention comprisenucleic acid aptamer based signaling polynucleotides (SPNs) that canspecifically bind to an allergen with high affinity. In otherembodiments, said SPN further comprises a fluorophore at one end of thenucleic acid sequence and a quencher at the opposite end. The SPNs ofthe present invention may comprise a polynucleotide sequence wherein 5to 20 nucleobase residues at the 5′-end of the sequence are at least 80%complementary to 5 to 20 nucleobase residues at the 3′-end of thesequence capable of forming a hairpin structure, thereby bringing thequencher in sufficient proximity to the fluorophore for quenching thefluorescence of the fluorophore.

In some embodiments, a SPN may comprise a polynucleotide sequenceselected from the sequences listed in Table 1 of the present disclosure.

In some embodiments, provided in the present invention are assays andmethods for detection of one or more allergens in a test sample. In someembodiments, the methods may comprise the steps of (a) obtaining a testsample suspected of containing an allergen; (b) processing the testsample and extracting proteins from the processed sample using anextraction buffer; (c) mixing the protein extraction of step (b) with aSPN that specifically binds to the allergen; (d) activating the SPN bymeans of an energy excitation; and (e) visualizing the interactionbetween the SPN and the allergen protein and detecting the absence orpresence of the allergen in the test sample. In some embodiments, totalproteins extracted from the test sample are determined and theextraction buffer is optimized for maximal protein extraction. In otherembodiments, the amount of the allergen presented in the test sample isdetermined.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the secondary sequence of a detection molecule representedby signaling polynucleotide SPN-A* 200 (SEQ ID NO. 2) which comprisecore sequence 202 (SEQ ID NO.1), fluorophore 204, quencher 206 andlinker sequence 208 (SEQ ID NO. 3).

FIG. 2 shows a reaction between a detection molecule represented by ahairpin-type signaling polynucleotide SPN-E 300 (SEQ ID NO. 7) with itstarget molecule lysozyme. Also hown are the aptamer core sequence 302(SEQ ID NO. 4), the fluorophore 304 and the quencher 306.

FIG. 3 shows a reaction between a detection molecule represented by adimeric signaling polynucleotide SPN-E* 400 (SEQ ID NO. 5) (including anannealed linker sequence 408 (SEQ ID NO. 6)) with its target moleculelysozyme. Also shown are the aptamer core sequence 402 (SEQ ID NO.4),the fluorophore 404 and the quencher 406.

DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Although any materials and methodssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred materialsand methods are now described. Other features, objects and advantages ofthe invention will be apparent from the description. In the description,the singular forms also include the plural unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. In the case of conflict, the present description will control.

Nucleic acid aptamers that can specifically bind to an allergen proteinwith high affinity are selected and signaling polynucleotides aredesigned using selected aptamers and tested for detection of an allergenin the present disclosure.

Allergens include those from food products, the environment or animalssuch as a domestic pet dander. Food allergens include, but are notlimited to proteins in legumes such as peanuts, peas, lentils and beans,tree nuts, wheat, milk, fish, egg white and sea food. Other allergensmay be from the environment such as pollens, other animals (e.g., pet),pathogens and medicines. A comprehensive list of allergenic proteinsfrom various sources is discussed below.

Composition of the Invention

Described herein are compositions, methods for the design, preparation,use and manufacture of the compositions, and methods and assays fordetection of a target protein(s) in a sample, in particular an allergenprotein(s).

Aptamers

In accordance with the present invention, compositions of the presentinvention include, but are not limited to any molecule or moleculeswhich are capable of association or binding to one or more allergens. Insome embodiments, compositions of the invention comprise one or moreaptamers.

As used herein, an “aptamer” is a nucleic acid species that has beenengineered through repeated rounds of in vitro selection orequivalently, SELEX (systematic evolution of ligands by exponentialenrichment) to bind to various molecular targets such as smallmolecules, proteins, nucleic acids, and even cells, tissues andorganisms. Nucleic acid aptamers have specific binding affinity tomolecules through interactions other than classic Watson-Crick basepairing. Nucleic acid aptamers, like peptides generated by phage displayor monoclonal antibodies (mAbs), are capable of specifically binding toselected targets and, through binding, block their targets' ability tofunction. In some cases, aptamers may also be peptide aptamers. As usedherein, an “aptamer” specifically refers to a nucleic acid aptamer.

Aptamers, often called “chemical antibodies,” have characteristics whichare similar to those of antibodies. A typical nucleic acid aptamer isapproximately 10-15 kDa in size (20-45 nucleotides), binds its targetwith at least nanomolar affinity, and discriminates against closelyrelated targets.

Nucleic acid aptamers may be either monovalent or multivalent. Aptamersmay be monomeric, dimeric, trimeric, tetrameric or higher multimeric.Individual aptamer monomers may be linked to form multimeric aptamerfusion molecules. As a non-limiting example, a linking oligonucleotide(i.e., linker) may be designed to contain sequences complementary toboth 5′-arm and 3′-arm regions of random aptamers to form dimericaptamers. For trimeric or tetrameric aptamers, a small trimeric ortetrameric (i.e., a Holiday junction-like) DNA nanostructure will beengineered to include sequences complementary to the 3′-arm region ofthe random aptamers, therefore creating multimeric aptamer fusionthrough hybridization. In addition, 3 to 5 or 5 to 10 dT richnucleotides can be engineered into the linker polynucleotides as asingle stranded region between the aptamer-binding motifs, which offersflexibility and freedom of multiple aptamers to coordinate and synergizemultivalent interactions with cellular ligands or receptors.

Alternatively, multimeric aptamers can also be formed by mixingbiotinylated aptamers with streptavidin.

As used herein, the term “multimeric aptamer” or “multivalent aptamer”refers to an aptamer that comprises multiple monomeric units, whereineach of the monomeric units can be an aptamer on its own. Multivalentaptamers have multivalent binding characteristics. A multimeric aptamercan be a homomultimer or a heteromultimer. The term “homomultimer”refers to a multimeric aptamer that comprises multiple binding units ofthe same kind, i.e., each unit binds to the same binding site of thesame target molecule. The term “heteromultimer” refers to a multimericaptamer that comprises multiple binding units of different kinds, i.e.,each binding unit binds to a different binding site of the same targetmolecule, or each binding unit binds to a binding site on differenttarget molecule. Thus, a heteromultimer can refer to a multimericaptamer that binds to one target molecule at different binding sties ora multimeric aptamer that binds to different target molecules. Aheteromultimer that binds to different target molecules can also bereferred to as a multi-specific multimer.

Nucleic acid aptamers comprise a series of linked nucleosides ornucleotides. The term “nucleic acid,” in its broadest sense, includesany compound and/or substance that comprise a polymer of nucleotides.These polymers are often referred to as polynucleotides. Exemplarynucleic acid molecules or polynucleotides of the invention include, butare not limited to, either D- or L-nucleic acids, ribonucleic acids(RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs),glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), lockednucleic acids (LNAs, including LNA having a β-D-ribo configuration,α-LNA having an α-L-ribo configuration (a diastereomer of LNA),2′-amino-LNA having a 2′-amino functionalization, and 2′-amino-α-LNAhaving a 2′-amino functionalization) or hybrids thereof.

The skilled artisan will recognize that the term “RNA molecule” or“ribonucleic acid molecule” encompasses not only RNA molecules asexpressed or found in nature, but also analogs and derivatives of RNAcomprising one or more ribonucleotide/ribonucleoside analogs orderivatives as described herein or as known in the art. Strictlyspeaking, a “ribonucleoside” includes a nucleoside base and a ribosesugar, and a “ribonucleotide” is a ribonucleoside with one, two or threephosphate moieties. However, the terms “ribonucleoside” and“ribonucleotide” can be considered to be equivalent as used herein. TheRNA can be modified in the nucleobase structure, the ribofuranosyl ringor in the ribose-phosphate backbone.

Nucleic acid aptamers may be ribonucleic acid, deoxyribonucleic acid, ormixed ribonucleic acid and deoxyribonucleic acid. Aptamers may be singlestranded ribonucleic acid, deoxyribonucleic acid or mixed ribonucleicacid and deoxyribonucleic acid.

In some embodiments, the aptamer comprises at least one chemicalmodification. In some embodiments, the chemical modification is selectedfrom a chemical substitution of the nucleic acid at a sugar position, achemical substitution at a phosphate position and a chemicalsubstitution at a base position. In other embodiments, the chemicalmodification is selected from incorporation of a modified nucleotide; 3′capping; conjugation to a high molecular weight, non-immunogeniccompound; conjugation to a lipophilic compound; and incorporation ofphosphorothioate into the phosphate backbone. In a preferred embodiment,the high molecular weight, non-immunogenic compound is polyalkyleneglycol, and more preferably is polyethylene glycol (PEG). The process ofcovalent conjugation of PEG to another molecule, normally a drug ortherapeutic protein is known as PEGylation. PEGylation is routinelyachieved by incubation of a reactive derivative of PEG with the targetmolecule. The covalent attachment of PEG to a drug or therapeuticprotein can mask the agent from the host's immune system, therebyproviding reduced immunogenicity and antigenicity, and increase thehydrodynamic size (size in solution) of the agent which prolongs itscirculatory time by reducing renal clearance. PEGylation can alsoprovide water solubility to hydrophobic drugs and proteins.

In another preferred embodiment, the 3′ cap is an inverteddeoxythymidine cap.

In some embodiments, nucleic acid aptamers are provided in which theP(O)O group is replaced by P(O)S (“thioate”), P(S)S (“dithioate”),P(O)NR2 (“amidate”), P(O)R, P(O)OR′, CO or CH2 (“formacetal”) or3′-amine (—NH—CH2-CH2-), wherein each R or R′ is independently H orsubstituted or unsubstituted alkyl. Linkage groups can be attached toadjacent nucleotide through an —O—, —N—, or -S-linkage. Not all linkagesin the nucleic acid aptamers are required to be identical.

As non-limiting examples, a nucleic acid aptamer can include D-ribose orL-ribose nucleic acid residues and can also include at least onemodified ribonucleoside including but not limited to a 2′-O-methylmodified nucleoside, a nucleoside comprising a 5′ phosphorothioategroup, a terminal nucleoside linked to a cholesteryl derivative ordodecanoic acid bisdecylamide group, a locked nucleoside, an abasicnucleoside, an inverted deoxynucleoside or inverted ribonucleoside, a2′-deoxy-2′-fluoro-modified nucleoside, a 2′-amino-modified nucleoside,a 2′-alkyl-modified nucleoside, a morpholino nucleoside, aphosphoramidate or a non-natural base comprising nucleoside, or anycombination thereof. Alternatively, a nucleic acid aptamer can compriseat least two modified ribonucleosides, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least15, at least 20 or more modified ribonucleosides, up to the entirelength of the molecule. The modifications need not be the same for eachof such a plurality of modified deoxy- or ribonucleosides in a nucleicacid molecule.

Detection molecules which are nucleic acid based may include nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines andguanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in Modified Nucleosides in Biochemistry, Biotechnology andMedicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. L, ed. John Wiley & Sons, 1990, those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Researchand Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRCPress, 1993.

A suitable nucleotide length for an aptamer ranges from about 15 toabout 100 nucleotides (nt), and in various other preferred embodiments,15-30 nt, 20-25 nt, 30-100 nt, 30-60 nt, 25-70 nt, 25-60 nt, 40-60 nt,25-40 nt, 30-40 nt, any of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nt or 40-70 nt inlength. In some embodiments, an aptamer may be 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, or 70 nt in length. In other embodiments, an aptamermay be 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nt in length.However, the sequence can be designed with sufficient flexibility suchthat it can accommodate interactions of aptamers with two targets at thedistances described herein.

In some embodiments, the nucleic acid aptamer comprises one or moreregions of double-stranded character. Such double stranded regions mayarise from internal self-complementarity or complementarity with asecond or further aptamers or oligonucleotide molecule. In someembodiments, the double stranded region may be from 4-12, 4-10, 4-8 basepairs in length. In some embodiments, the double stranded region may be5, 6, 7, 8, 9, 10, 11 or 12 base pairs. In some embodiments, the doublestranded region may form a stem region. Such extended stem regionshaving double stranded character can serve to stabilize the nucleic acidaptamer. As used herein, the term “double stranded character” means thatover any length of two nucleic acid molecules, their sequences form basepairings (standard or nonstandard) of more than 50 percent of thelength.

Aptamers may be further modified to provide protection from nuclease andother enzymatic activities. The aptamer sequence can be modified by anysuitable methods known in the art. For example, phosphorothioate can beincorporated into the backbone, and 5′-modified pyrimidine can beincluded in 5′ end of ssDNA for DNA aptamers. For RNA aptamers, modifiednucleotides such as substitutions of the 2′-OH groups of the ribosebackbone, e.g., with 2′-deoxy-NTP or 2′-fluoro-NTP, can be incorporatedinto the RNA molecule using T7 RNA polymerase mutants. The resistance ofthese modified aptamers to nuclease can be tested by incubating themwith either purified nucleases or nuclease from mouse serum, and theintegrity of aptamers can be analyzed by gel electrophoresis.

In some embodiments, such modified nucleic acid aptamers may besynthesized entirely of modified nucleotides, or with a subset ofmodified nucleotides. The modifications can be the same or different.All nucleotides may be modified, and all may contain the samemodification. All nucleotides may be modified, but contain differentmodifications, e.g., all nucleotides containing the same base may haveone type of modification, while nucleotides containing other bases mayhave different types of modifications. For example, all purinenucleotides may have one type of modification (or are unmodified), whileall pyrimidine nucleotides have another, different type of modification(or are unmodified). In this way, oligonucleotides, or libraries ofoligonucleotides are generated using any combination of modifications asdisclosed herein.

According to certain embodiments of the present invention, variants andderivatives of aptamers are provided. The term “derivative” is usedsynonymously with the term “variant” and refers to a molecule that hasbeen modified or changed in any way relative to a reference or startingaptamer. The nucleic acid sequence of aptamer variants may possesssubstitutions, deletions, and/or insertions at certain positions withinthe nucleotide sequence, as compared to a reference or startingsequence. Ordinarily, variants will possess at least about 50% identity(homology) to a reference sequence, and preferably, they will be atleast about 80%, more preferably at least about 90% identical(homologous) to a reference sequence.

In some embodiments, variant mimics of aptamers of the present inventionare provided. As used herein, the term “variant mimic” is one whichcontains one or more nucleic acids which would mimic an activatedsequence. The nucleic acid sequences of variant mimics may comprisenaturally occurring nucleic acids, or alternatively, non-naturallyoccurring nucleic acids.

Aptamer Conjugates and Labels

In some embodiments, aptamers of the invention may comprise conjugates.Such conjugates of the invention may include a naturally occurringsubstance or ligand, such as a protein; a carbohydrate (e.g., a dextran,pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); ora lipid; as well as a recombinant or synthetic molecule, such as asynthetic polymer.

Examples of conjugates may include, but are not limited to magneticnanoparticles (MNPs) (e.g., superparamagnetic Iron Oxide Nanoparticles(SPIONs), gold NPs, and quantum dots (QDs)); chitosans; and drugconjugates.

In some embodiments, aptamers of the present invention may comprise adetectable agent, such as various organic small molecules, inorganiccompounds, nanoparticles, enzymes or enzyme substrates, fluorescentmaterials, luminescent materials (e.g., luminol), bioluminescentmaterials (e.g., luciferase, luciferin, and aequorin), chemiluminescentmaterials, radioactive materials (e.g., 18F, 67Ga, 81mKr, 82Rb, 111In,1231, 133Xe, 201Tl, 125I, 35S, 14C, 3H, or 99mTc (e.g., as pertechnetate(technetate(VII), TcO4)), and contrast agents (e.g., gold (e.g., goldnanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g.,superparamagnetic iron oxide (SPIO), monocrystalline iron oxidenanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide(USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinatedcontrast media (iohexol), microbubbles, or perfluorocarbons). Suchoptically-detectable labels include for example, without limitation,4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid; acridine andderivatives (e.g., acridine and acridine isothiocyanate);5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives (e.g., coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120), and7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives (e.g., eosin and eosin isothiocyanate); erythrosin andderivatives (e.g., erythrosin B and erythrosin isothiocyanate);ethidium; fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM),dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, X-rhodamine-5-(and-6)-isothiocyanate (QFITCor XRITC), and fluorescamine);2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indoliumhydroxide, inner salt, compound with N,N-diethylethanamine(1:1) (IR144);5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140); Malachite Green isothiocyanate;4-methylumbelliferone orthocresolphthalein; nitrotyrosine;pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyreneand derivatives (e.g., pyrene, pyrene butyrate, and succinimidyl1-pyrene); butyrate quantum dots; Reactive Red 4 (CIBACRON™ BrilliantRed 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodanine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolicacid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; LaJolta Blue; phthalo cyanine; and naphthalo cyanine.

In some embodiments, the detectable agent may be a non-detectablepre-cursor that becomes detectable upon activation (e.g., fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL,tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzymeactivatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical))). Insome embodiments, the non-detectable precursor comprises a combinationof a fluorophore and a quencher, such as the combination of fluoresceinand DABCYL, for example. Guidelines for selection of fluorophore andquencher pairs are described in S.A.E. Marras Selection of Fluorophoreand Quencher Pairs for Fluorescent Nucleic Acid Hybridization Probes inDidenko, Vladimir V., ed. Fluorescent energy transfer nucleic acidprobes: designs and protocols. Vol. 335. Springer, 2006, which isincorporated herein by reference in entirety.

Signaling Polynucleotides (SPNs)

In accordance with certain embodiments, there are providedpolynucleotide sequences that are detectable when bound at high affinityand specificity to allergen targets. Such polynucleotide sequences maybe produced using the SELEX process as described hereinabove.

In certain types of exemplary signaling polynucleotides, the 5′ end ofthe sequence is bound to a fluorescent molecule and the 3′ end carries a5-20 nucleotide long reverse-complement sequence that binds to the5′-end. This results of folding of the sequence and formation of astem-loop structure. A quencher molecule is bound to the 3′-end. Theskilled person will recognize that alternative arrangements are possiblewherein the quencher is bound to the 5′-end and the fluorophore is boundto the 3′-end. Such alternative signaling polynucleotides may beprepared by the skilled person in context of the present descriptionwithout undue experimentation.

An exemplary signaling polynucleotide designed with a stem-loopstructure for binding to lysozyme as a molecular target will bedescribed herein below in Example 1.

In certain embodiments, the fluorophore molecule at the 5′-end is boundto a T nucleotide residue in order to prevent quenching caused by a Gnucleotide residue.

In recognition that higher melting temperatures (Tm) are to be avoided,the Tm or ΔG of the two strands will need to be lower than the bindingaffinity of the molecular target in order for the signalingpolynucleotide to have a thermodynamic preference for binding to themolecular target. In order to retain preferable molecular targetbinding, Mg⁺² or K⁺ may be added to shift the equilibrium. Addition ofup to about 37 mM KCl will shift the equilibrium of a given signalingpolynucleotide to favor binding of a molecular target while adding up toabout 5 mM MgCl₂ will shift the equilibrium towards retention of thedouble strand structure, thereby lowering the affinity of the signalingpolynucleotide for its molecular target.

It is not necessary for the two reverse complementary strands to be onopposite sides in order to create a stem-loop structure. The reversecomplementary strand can be attached/annealed to the 5′-end. Thesequence must be long enough to physically interfere with the structure.The double strand binding needs to prevent the formation of thesecondary structure folding which is needed in order to bind themolecular target.

In certain embodiments, the signaling polynucleotides are dimericentities with a core sequence linked to a fluorophore and a shorterannealed linker sequence linked to a quencher, or vice versa. In oneexample, the signaling polynucleotide may comprise a linker sequence 5to 20 nucleobases in length annealed to the 5′-end of the sequence ofthe signaling polynucleotide and having at least 80% complementaritywith the 5′-end of the sequence of the signaling polynucleotide, whereinthe signaling polynucleotide comprises a fluorophore and the linkersequence comprises a quencher.

In certain embodiments, the signaling polynucleotide sequences arechemically modified with 2′-O-methyl modifications. Such modificationsare expected to not significantly affect the binding affinity andsensitivity with respect to binding of the molecular target, whileenhancing stability.

In some embodiments, signaling polynucleotides against several commonfood allergens are designed using polynucleotides (e.g., aptamers)selected from SELEX processes as described above herein. The nucleicacid sequences of such aptamers have high binding affinity andspecificity to the allergens. Table 1 lists the aptamer sequences fromwhich the signaling polynucleotides are designed.

TABLE 1 SPNs that bind to food allergens SEQ Reference ID Allergen No.Sequence (5′-3′) NO. Peanut MB4 {FAM}TTCGCGCACATTCCGCTTCTACCGGGGGGG 8TCGAGCTGAGTGGATGCGAATCTGTGGGTGGGCC GTAAGTCCGTGTGTGCGAA{DABCYL} PeanutMB7 {FAM}TCGCACATTCCGCTTCTACCGGGGGGGTCG 9AGCTGAGTGGATGCGAATCTGTGGGTGGGCCGTA AGTCCGTGTGTGCGAAAATGTGCGA{DABCYL}Peanut MB9 {FAM}TCGCACATTCCGCTTCTACCGGGGGGGTCG 10AGCTGAGTGGATGCGAATCTGTGGGTGGGCCGTA AGTCCGTGTGTGCGAATGTGCGA{DABCYL} EggE-MB7 {FAM}TGGCAGCTAAGCAGGCGGCTCACAAAACC 11 whiteATTCGCATGCGGCTGTTCCA{DABCYL} Egg E-MB6{FAM}TGCAGCTAAGCAGGCGGCTCACAAAACCA 12 white TTCGCATGCGGCGCTGCA{DABCYL}Egg E-MB4 {FAM}TGCAGCTAAGCAGGCGGCTCACAAAACCA 13 whiteTTCGCATGCGGCTGCA{DABCYL} Egg E-MB5 {FAM}GCAGCTAAGCAGGCGGCTCACAAAACCAT 14white TCGCATGCGGCGCTGC{DABCYL} Wheat Gli4 + 4{FAM}TTTCCCAGTCTCCCGTTTACCGCGCCTACA 15 CATGTCTGAATGCCGAAA{DABCYL} WheatGL1_7_4 {FAM}TCGAAAAGCTGCAGCTGCAACCATTTCCGC 16AGCCGCAACTACCATATCCGCAGCCGCAACTACC ATATCCGCAGCCGCAACTACCATATCCGCAGCGGCAACCATTTTCGA{DABCYL} Wheat G33_16_1/2{FAM}AACAAACTACTAACTAGGTAAGATCACGC 17AGCACTAAACGACGTAGTTGCCATGTT{DABCYL} Wheat G33_7{FAM}TGGCAAACTACTAACTAGGTAAGATCACG 18 CAGCACTAAACGACGTAGTTGCCA{DABCYL}Wheat G33_14_1/2 {FAM}TTGGAAACTACTAACTAGGTAAGATCACG 19CAGCACTAAACGACGTAGTTGCCAA{DABCYL} Wheat S_Gluten_11_7{FAM}CCGAGCTAAATGCTGCAGCTGCAACCATTT 20 1/2CCGCAGCCGCAACTACCATATCCGCAGCCGCAAC TACCATATCCGCAGCCGCAACTACCATATCCGCAGCGGCAACCATTTAGCTCGG{DABCYL} Wheat S_Gluten_8_4 1{FAM}CCGAAAATGCTGCAGCTGCAACCATTTCCG 21CAGCCGCAACTACCATATCCGCAGCCGCAACTAC CATATCCGCAGCCGCAACTACCATATCCGCAGCGGCAACCATTTTCGG{DABCYL} Wheat GLI_6 1/2/3{FAM}CCAGTCTCCCGTTTACCGCGCCTACACATG 22 TCTGAATGCCGACTGG{DABCYL} WheatGLI_4_1/2 {FAM}GGCACCAGTCTCCCGTTTACCGCGCCTACA 23 CATGTCTGAATGCC{DABCYL}Milk 457_12 {FAM}AUGAGCUUGGUCACCUUUCCUGACAUUA 24ACACAGGCGAAACGGUGAAAGCCGU{DABCYL} Milk 491_5E/F{FAM}CAUGAGUUUUCCCGAUACGGCUACGAAU 25 UGCGACAACGAAACGGUGAAAGCCGUG{DABCYL} Milk 491_2_11 {FAM}UGAGUUUUCCCGAUACGGCUACGAAUUG 26CGACAACGAAACGGUGAAAGCCCA{DABCYL}

In addition to the nucleic acid aptamers selected from the SELEX processas described in Examples. Signaling polynucleotides may be designedusing aptamers selected from other studies. The 5′-end and 3′endnucleotides, fluorophores/quencher pairs and the stem-loop structuresmay be further designed according to the criteria described above, andtested for their binding affinity and specificity to the target.

In some embodiments, SPNs may be developed using aptamers against foodallergens as disclosed in the art. Such aptamers may include, but arenot limited to, aptamers specific to Lup an 1 (β-conglutin) (Nadal P etal., DNA Aptamers against the Lup an 1 Food Allergen, PLos One, 2012, 7:e35253); leptin (lep3) (Ashley and Li, Three-dimensional selection ofleptin aptamers using capillary electrophoresis and implications forclone validation, Anal Biochem., 2013, 434: 146-152); and lysozyme (eggwhite) (Kirby et al., Aptamer-based sensor arrays for the detection andquantitation of proteins, Anal Chem. 2004, 76(14): 4066-4075; Zou M etal., The homogeneous fluorescence anisotropic sensing of salivarylysozyme using the 6-carboxyfluorescein-labeled DNA aptamer, BiosensBioelectron, 2012, 32(1): 148-154; Robertson and Ellington, In vitroselection of nucleootein enzymes, Nature Biotechnology, 2001: 650-655;and Hesselberth et al, Simultaneous detection of diverse analytes withan aptazyme ligase array, Analytical Biochemistry, 2003, 312: 106-112;each of which is incorporated herein by reference in its entirety.) Thenucleic acid sequences of the aptamers from each disclosure are listedin Table 2.

In some embodiments, signaling polynucleotides may be developed usingaptamers that bind to Cry j 2 allergen of Japanese cedar pollen (Ogiharaet al., DNA aptamers against Cry j 2 allergen of Japanese cedar pollenfor biosensing applications, Biosens Bioelectron., 2015, 63, 159-165),lup an 1 (β-conglutin) subunit present in lupine flour (Svobodova etal., Ultrasensitive aptamer based detection of β-conglutin foodallergen, Food Chem., 2014, 165, 419-423; and Mairal et al., FRET-baseddimeric aptamer probe for selective and sensitive Lup an 1 allergendetection, Biosens Bioelectron., 2014, 54: 207-210), and gliadin(gluten) (Pinto A et al., Label-free detection of gliadin food allergenmediated by real-time apta-PCR, Anal Bioanal Chem., 2014, 406(2):515-524); each of which is incorporated herein by reference in itsentirety.

TABLE 2 Aptamers against food allergens SEQ ID Allergen ReferenceSequence (5′-3′) NO. Lup an 1 Nadal et al.,AGCTGACACAGCAGGTTGGTGGGGGTGGCTTCC 27 Plos One,AGTTGGGTTGACAATACGTAGGGACACGAAGT 2012, 7: CCAACCACGAGTCGAGCAATCTdCGApAATe35253 Lup an 1 Nadal et al., GGTGGGGGTGG 28 Anal. Bioanal. Chem., 2013,405: 9343-9349 Lep Ashley and Li, CTTCTGCCCGCCTCCTTCCGTTAATGGGGGATCT 293(leptin) Anal CGCGGCCGTTCTTGTTGCTTATACAGGAGACGA Biochem.,GATAGGCGGACACT 2013, 434: 146-152 Lysozyme Kirby et al.,GGGAATGGATCCACATCTACGAATTCATCAGGG 30 (Egg Anal Chem.CTAAAGAGTGCAGAGTTACTTAGTTCACTGCAG white) 2004, 76(14): ACTTGACGAAGCTT4066-4075 Lysozyme Zou M et al., AGCAGCACAGAGGTCAGATGGCAGGTAAGCAG 31(Egg Biosens GCGGCTCACAAAACCATTpdCGCATGCGGCCCT white) Bioelectron,ATGCGTGCTACCGTGAA 2012, 32(1): 148-154 Lysozyme Robertson andrGGAprCCUprCGGprCGAprAAGprCprUAAprCGUpr 32 (Egg Ellington,CUCprAUGprGCUprAAAprUUGprCCAprUGUprUG white) 2001, NatureCprUACprAAAprUGAprUAUprGACprUAGprAprGA Biotech.,GprGUUprAGGprUGCprCUCprGUGprAUGprUCCpr 2001, 650-655 AGUprCGCp

In some embodiments, signaling polynucleotides may be developed usingaptamers that are selected as detection molecules for pathogens. Asnon-limiting examples, aptamers that can specifically recognizeSalmonella, Listeria, E. coli, and aspergillus fumigatus may be used todesign signaling polynucleotides (SPNs) as described herein. Suchaptamers are discussed in, for example: Han and Lee, In Vitro Selectionof RNA Aptamer Specific to Salmonella Typhimurium, Journal ofMicrobiology and Biotechnology, 2013, 23: 878-884; Hyeon, J. et al.Development of RNA Aptamers for Detection of Salmonellas Enteritidis,Journal of Microbiological Methods, 2012, 89:79-82; Ohk et al.,Antibody-aptamer functionalized fibre-optic biosensor for specificdetection of Listeria monocytogenes from food, J. Appl. Microbiol.,2010, 109: 808-817; Li, H. et al., Aptamer selection for the detectionof Escherichia coli K88, Canadian Journal of Microbiology, 2011, 57:453-459; Lee at al., In vitro selection of Escherichia coliO157:H7-specific RNA aptamer, Biochemical and Biophysical ResearchCommunications, 2012, 417: 214-220; Ali et al., Fluorogenic DNAzyme obesas Bacterial Indicators, Angewandte Chemie International Edition, 2011,50: 3751-3754; and DeGrasse J A, A Single-Stranded DNA Aptamer ThatSelectively Binds to Staphylococcus aureus Enterotoxin B, Plos One,2012, 7: e33410; each of which is incorporated herein by reference inits entirety. Table 3 lists the sequences of such aptamers from thosedisclosures.

TABLE 3 Aptamers against pathogens SEQ ID Pathogen ReferenceSequence (5′-3′) NO. Salmonella Han and Lee, J.fUAGfUGfUGAGAGfCCGfUGAGfUGAAAGGf 33 Microbio. &CfCGfCGAfCAAAGAfUfCGGA ((f = 2′-f- Biotech., 2013, pyrimidines) 878-884Salmonella Hyeon, J. et al., J. GGGUUCACUGCAGACUUGACGAAGCUUG 34Enteritidis Microbio. AGAGAUGCCCCCUGAUGpUGCAUUCUUG Methods, 2012,UUGUGUUGCGGCAAUGGAUCCACAUCUA 79-82 CGAAUUC Listeria Ohk et al., J.ATCCATGGGGCGGAGATGAGGGGGAG 35 Appl. Microbiol., GAGGGCGGGTACCCGGTTGAT2010, 109: 808-817 E. coli Li et al., GGAGACCGTACCATCTGTTCGTGGAAGCG 36fimbriae Canadian CTTTGCTCGTCCATTAGCCTTGTGCTCGTGC otein K88 Journal ofMicrobiology, 2011, 57: 453-459. E. coli Lee et al.,GGGfUfCfUfUfCfCfUGGAfCfUGfUfCGAAAA 37 O157:H7 BiophysicalfUfUfCAGfUAfUfCGGGAGGfUfUAfCGfUAfU ResearchfUfUGGfUfUfUAfUAGAfUAGfUAA (f = 2′-f- Communications, pyrimidines)2012, 417: 214-220 E. Coli Ali et al., CACGGATCCTGACAAGGATGTGTGCGTTG 38mixture Angewandte TCGAGACCTGCGACCGGAACACTACACTG ChemieTGTGGGATGGATTTCTTTACAGTTGTGTGC International AGCTCCGTCCGACT CTTCCTAGC-Edition, 2011, {Internal/Fluorescein-dT}-Aptamer- 50: 3751-3754{Internal/Dabcyl-dT}-GGTTCGATCAAGA Staphylococcus DeGrasse JA,GGTATTGAGGGTCGCATCCACTGGTCG 39 aureus Plos One, 2012,TTGTTGTCTGTTGTCTGTTATGTTGTTTCG (enterotoxin 7: e33410TGATGGCTCTAACTCTCCTCT B)

In other embodiments, as a skilled artisan would envision, aptamers thatspecifically bind to non-protein targets, for example, a small moleculemay also be used to design signaling polynucleotides as disclosedherein. Table 4 lists the sequences of some aptamers as non-limitingexamples (Ferguson et al., A novel strategy for selection of allostericribozymes yields RiboReporter™ sensors for caffeine and aspartame,Nucleic Acids Research, 2004, 32: 1756-1766; and Ono and Togashi, Highlyselective oligonucleotide-based sensor for mercury(II) in aqueoussolutions, Angew. Chem. Int. Ed., 2004, 43: 4300-4302).

TABLE 4 Aptamers against non-protein targets SEQ Non protein ID targetReference Sequence (5′-3′) NO. Caffeine Ferguson et al.,GGAUGUCCAGUCGCUUGCAAUGCCCUUU 40 Nucleic AcidsUAGACCCUGAUGAGGAUCAUCGGACUUU Research, 2004,GUCCUGUGGAGUAAGAUCGCGAAACGGU 32: 1756-1766 GAAAGCCGUAGGUCU Mercury IIOno and Togashi, TTCTTTCTTCCCCTTGTTTGTT 41 (Hg2+) Angew. Chem. Int. Ed.,2004, 43: 4300-4302Targets of the Signaling Polynucleotides

The present invention provides aptamer based signaling polynucleotides(SPNs) that bind to a target molecule. As stated below, the targetmolecule may be an allergen protein or variants thereof. In someembodiments, SPNs may be designed to bind or associate with proteins orother biomolecules which themselves associated with the allergen.

According to the present invention, and while not wishing to be bound bytheory, the detection polynucleotides may completely or partially bindan allergen.

Allergens

In some embodiments, allergens are food allergens. Examples ofallergenic proteins associated with food include, but are not limitedto, Brine shrimp (Art fr 5), Crab (Cha f 1), North Sea Shrimp (Cra c 1,Cra c 2, Cra c 4, Cra c 5, Cra c 6, Cra c 8), American lobster (Hom a 1,Hom a 3, Hom a 6), white shrimp (Lit v 1, Lit v 2, Lit v 3, Lit v4),giant freshwater prawn (Mac r 1), shrimp (Met e 1, Pen a 1, Pen i 1),northern shrimp (Pan b 1), spiny lobster (Pan s 1), black tiger shrimp(Pen m 1, Pen m 2, Pen m 3, Pen m 4, Pen m 6), narrow-clawed crayfish(Pon i 4, Pon i 7), blue swimmer crab (Por p 1), domestic cattle (Bos d4, Bos d 5, Bos d 6, Bos d 7, Bos d 8, Bos d 9, Bos d 10, Bos d 11, Bosd 12), Atlantic herring (Clu h 1), common carp (Cyp c 1), Baltic cod(Gad c 1), Atlantic cod (Gad m 1, Gad m 2, Gad m 3), cod (Gad c 1),chicken (Gal d 1, Gal d 2, Gal d 3, Gal d 4, Gal d 5), Barramunda (Lat c1), Lepidorhombus whiffiagonis (Lep w 1), chum salmon (Onc k 5),Atlantic salmon (Sal s 1, Sal s 2, Sal s 3) rainbow trout (Onc m 1),Mozambique tilapia (Ore m 4), edible frog (Ran e 1, Ran e 2), pacificpilchard (Sar sa 1), ocean perch (Seb m 1), yellowfin tuna (Thu a 1, Thua 2, Thu a 3), swordfish (Xip g 1), abalone (Hal m 1), brown gardensnail (Hel as 1), Squid (Tod p 1), pineapple (Ana c 1, Ana c 2),asparagus (Aspa o 1), barley (Hor v 12, Hor v 15, Hor v 16, Hor v 17,Hor v 20, Hor v 21), banana (Mus a 1, Mus a 2, Mus a 3, Mus a 4, Mus a5), banana (Musxpl), rice (Ory s 12), rye (Sec c 20), wheat (Tri a 12,Tri a 14, Tri a 18, Tri a 19, Tri a 25, Tri a 26, Tri a 36, Tri a 37),maize (corn) (Zea m 14, Zea m 25), kiwi fruit (Act c1, Act c 2, Act c 5,Act c 8, Act c 10, Act d 1, Act d 2, Act d 3, Act d 4, Act d 5, Act d 6,Act d 7, Act d 8, Act d 9, Act d 10, Act d 11), cashew (Ana o 1, Ana o2, Ana o 3), celery (Api g 1, Api g 2, Api g 3, Api g 4, Api g 5, Api g6), peanut (Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h7, Ara h 8, Ara h 9, Ara h 10, Ara h 11, Ara h 12, Ara h 13), brazil nut(Ber e 1, Ber e 2), oriental mustard (Bra j 1), rapeseed (Bra n 1),cabbage (Bra o 3), turnip (Bra r 1, Bra r 2), bell pepper (Cap a 1w, Capa 2), pecan (Car i 1, Car i 4), chestnut (Cas s 1, Cas s 5, Cas s 8, Cass 9), lemon (Cit I 3), tangerine (Cit r 3), sweet orange (Cit s 1, Cit s2, Cit s 3), Hazel (Cor a 1, Cor a 2, Cor a 8, Cor a 9, Cor a 11, Cor a12, Cor a 13, Cor a 14), muskmelon (Cuc m 1, Cuc m 2, Cuc m 3), carrot(Dau c 1, Dau c 4, Dau c 5), common buckwheat (Fag e 2, Fag e 3),tartarian buckwheat (Fag t 2), strawberry (Fra a 1, Fra a 3, Fra a 4),soybean (Gly m 1, Gly m 2, Gly m 3, Gly m 4, Gly m 5, Gly m 6, Gly m 7,Gly m 8), sunflower (Hel a1, Hel a 2, Hel a 3), black walnut (Jug n 1,Jug n 2), English walnut (Jug r 1, Jug r 2, Jug r 3, Jug r 4),Cultivated lettuce (Lac s 1), Lentil (Len c 1, Len c 2, Len c 3), litchi(Lit c 1), narrow-leaved blue lupin (Lup an 1), apple (Mal d 1, Mal d 2,Mal d 3, Mal d 4), Cassava (Man e 5), mulberry (Mor n 3), avocado (Persa 1), green bean (Pha v 3), pistachio (Pis v 1, Pis v 2, Pis v 3, Pis v4, Pis v 5), pea (Pis s 1, Pis s 2), apricot (Pm ar 1, Pm ar 3), sweetcherry (Pm av 1, Pm av 2, Pm av 3, Pm av 4), European plum (Pm d 3),almond (Pm du 3, Pm du 4, Pm du 5, Pm du 6), peach (Pm p 1, Pm p 2, Pm p3, Pm p 4, Pm p 7), pomegranate (Pun g 1), pear (Pyr c 1, Pyr c 3, Pyr c4, Pyr c 5), castor bean (Ric c 1), red raspberry (Rub i 1, Rub i 3),Sesame (Ses i 1, Ses i 2, Ses i 3, Ses i 4, Ses i 5, Ses i 6, Ses i 7),yellow mustard (Sin a 1, Sin a 2, Sin a 3, Sin a 4), tomato (Sola I 1,Sola I 2, Sola I 3, Sola I 4), potato (Sola t 1, Sola t 2, Sola t 3,Sola t 4), Mung bean (Vig r 1, Vig r 2, Vig r 3, Vig r 4, Vig r 5, Vig r6), grape (Vit v 1), Chinese date (Ziz m 1), Anacardium occidentale (Anao 1.0101, Ana o 1.0102), Apium graveolens (Api g 1.0101, Api g 1.0201),Daucus carota (Dau c1.0101, Dau c1.0102, Dau c1.0103, Dau c1.0104, Dauc1.0105, Dau c1.0201), Citrus sinensis (Cit s3.0101, Cit s3.0102),Glycine max (Gly m1.0101, Gly m1.0102, Gly m3.0101, Gly m3.0102), Lensculinaris (Len c1.0101, Len c1.0102, Len c1.0103), Pisum sativum (Piss1.0101, Pis s1.0102), Lycopersicon sativum (Lyc e2.0101, Lyc e2.0102),Fragaria ananassa (Fra a3.0101, Fra a3.0102, Fra a3.0201, Fra a3.0202,Fra a3.0203, Fra a3.0204, Fra a3.0301), Malus domestica (Mal d1.0101,Mal d1.0102, Mal d1.0103, Mal d1.0104, Mal d1.0105, Mal d1.0106, Mald1.0107, Mal d1.0108, Mal d1.0109, Mal d1.0201, Mal d1.0202, Mald1.0203, Mal d1.0204, Mal d1.0205, Mal d1.0206, Mal d1.0207, Mald1.0208, Mal d1.0301, Mal d1.0302, Mal d1.0303, Mal d1.0304, Mald1.0401, Mal d1.0402, Mal d1.0403, Mal d3.0101w, Mal d3.0102w, Mald3.0201w, Mal d3.0202w, Mal d3.0203w, Mal d4.0101, Mal d4.0102, Mald4.0201, Mal d4.0202, Mal d4.0301, Mal d4.0302), Prunus avium (Pmav1.0101, Pm av1.0201, Pm av1.0202, Pm av1.0203), and Prunus persica (Pmp4.0101, Pm p4.0201); and any variants thereof. The names of allergensassociated with food are systematically named and listed according toIUIS Allergen Nomenclature Sub-Committee (see, International Union ofImmunological Societies Allergen Nomenclature Sub-Committee, List ofisoallergens and variants.)

In addition to food allergens, signaling polynucleotides of the presentinvention may detect airborne particulates/allergens and otherenvironmental allergens. Samples that contain allergens may be obtainedfrom plants (e.g. weeds, grasses, trees, pollens), animals (e.g.,allergens found in the dander, urine, saliva, blood or other bodilyfluid of mammals such as cat, dog, cow, pig, sheep, horse, rabbit, rat,guinea pig, mouse and gerbil), fungi/mold, insects (e.g., stinginginsects such as bee, wasp, and hornet and chirnomidae (non-bitingmidges), as well as other insects such as the housefly, fruit fly, sheepblow fly, screw worm fly, grain weevil, silkworm, honeybee, non-bitingmidge larvae, bee moth larvae, mealworm, cockroach and larvae ofTenibrio molitor beetle; spiders and mites such as the house dust mite),rubbers (e.g. latex), metals, chemicals (e.g. drugs, protein detergentadditives) and autoallergens and human autoallergens (e.g. Hom s 1, Homs 2, Hom s 3, Hom s 4, Hom s 5) (see, Allergen Nomenclature:International Union of Immunological Societies Allergen NomenclatureSub-Committee, List of allergens and Allergen Nomenclature:International Union of Immunological Societies Allergen NomenclatureSub-Committee, List of isoallergens and variants).

Examples of allergenic proteins from plants that can be detected usingthe compositions of the present invention include, but are not limitedto, ash (Fra e 1), Japanese cypress (Cha o1, Cha o 2), sugi (Cry j 1,Cry j 2), cypress (Cup a 1), common cypress (Cups 1, Cup s 3), mountaincedar (Jun a 1, Jun a 2, Jun a 3, Jun s 1), prickly juniper (Jun o 4),eastern red cedar (Jun v 1, Jun v 3), sweet vernal grass (Ant o 1),saffron crocus (Cro s 1, Cro s 2), Bermuda grass (Cyn d 1, Cyn d 7, Cynd 12, Cyn d 15, Cyn d 22w, Cyn d 23, Cyn d 24), orchard grass (Dac g 1,Dac g 2, Dac g 3, Dac g 4, Dac g 5), meadow fescue (Fes p 4), velvetgrass (Hol I 1, Hol I 5), barley (Hor v 1, Hor v 5), rye grass (Lol p 1,Lol p 2, Lol p 3, Lol p 4, Lol p 11), bahia grass (Pas n 1), canarygrass (Pha a 1, Pha a 5), timothy (Phl p 1, Phl p 2, Phl p 4, Phl p 5,Phl p 6, Phl p 7, Phl p 11, Phl p 12, Phl p 13), date palm (Pho d 2),Kentucky blue grass (Poa p 1, Poa p 5), rye (Sec c 1, Sec c 5, Sec c38), Johnson grass (Sor h 1), wheat (Tri a 15, Tri a 21, Tri a 27, Tri a28, Tri a 29, Tri a 30, Tri a 31, Tri a 32, Tri a 33, Tri a 34, Tri a35, Tri a 39), maize (Zea m 1, Zea m 12), alder (Aln g 1, Aln g 4),redroot pigweed (Ama r 2), short ragweed (Amba 1, Amba 2, Amba 3, Amba4, Amba 5, Amba 6, Amba 7, Amba 8, Amba 9, Amb a 10, Amb a 11), westernragweed (Amb p 5), giant ragweed (Amb t 5), mugwort (Art v 1, Art v 2,Art v 3, Art v 4, Art v 5, Art v 6), sugar beet (Beta v 1, beta v 2),European white birch (Bet v 1, Bet v 2, Bet v 3, Bet v 4, Bet v 6, Bet v7), turnip (Bra r 5), hornbeam (Car b 1), chestnut (Cas s 1), rosyperiwinkle (Cat r 1), lamb's-quarters, pigweed (Che a 1, Che a 2, Che a3), Arabian coffee (Cof a 1, Cof a 2, Cof a 3), Hazel (Cor a 6, Cor a10), Hazel nut (Cor a1.04, Cor a2, Cor a8), European beech (Fag s 1),ash (Fra e 1), sunflower (Hel a 1, Hel a 2), para rubber tree (Hev b 1,Hev b 2, Hev b 3, Hev b 4, Hev b 5, Hev b 6, Hev b 7, Hev b 8, Hev b 9,Hev b 10, Hev b 11, Hev b 12, Hev b 13, Hev b 14), Japanese hop (Hum j1), privet (Lig v 1), Mercurialis annua (Mer a 1), olive (Ole e 1, Ole e2, Ole e 3, Ole e 4, Ole e 5, Ole e 6, Ole e 7, Ole e 8, Ole e 9, Ole e10, Ole e 11), European hophornbeam (Ost c 1), Parietaria judaica (Par j1, Par j 2, Par j 3, Par j 4), Parietaria officinalis (Par o 1),Plantago lanceolata (Pal I 1), London plane tree (Pla a 1, Pla a 2, Plaa 3), Platanus orientalis (Pla or 1, Pla or 2, Pla or 3), white oak (Quea 1), Russian thistle (Sal k 1, Sal k 2, Sal k 3, Sal k 4, Sal k 5),tomato (Sola I 5), Lilac (Syr v 1, Syr v 5), Russian-thistle (Sal k 1),English plantain (Pla 11), Ambrosia artemisiifolia (Amb a8.0101, Amba8.0102, Amb a9.0101, Amb a9.0102), Plantago lanceolata (Pla l1.0101,Pla l1.0102, Pla l1.0103), Parietaria judaica (Par j 3.0102), Cynodondactylon (Cyn d1.0101, Cyn d1.0102, Cyn d1.0103, Cyn d1.0104, Cynd1.0105, Cyn d1.0106, Cyn d1.0107, Cyn d1.0201, Cyn d1.0202, Cynd1.0203, Cyn d1.0204), Holcus lanatus (Hol I1.0101, Hol I1.0102), Loliumperenne (Phl p1.0101, Phl p1.0102, Phl p4.0101, Phl p4.0201, Phlp5.0101, Phl p5.0102, Phl p5.0103, Phl p5.0104, Phl p5.0105, Phlp5.0106, Phl p5.0107, Phl p5.0108, Phl p5.0201, Phl p5.0202), Secalecereale (Sec c20.0101, Sec c20.0201), Betula Verrucosa (Bet v1.0101, Betv1.0102, Bet v 1.0103, Bet v 1.0201, Bet v 1.0301, Bet v1.0401, Bet v1.0402, Bet v 1.0501, Bet v 1.0601, Bet v 1.0602, Bet v1.0701, Betv1.0801, Bet v1.0901, Bet v1.1001, Bet v1.1101, Bet v1.1201, Bet v1.1301, Bet v1.1401, Bet v1.1402, Bet v1.1501, Bet v1.1502, Bet v1.1601,Bet v1.1701, Bet v 1.1801, Bet v1.1901, Bet v1.2001, Bet v1.2101, Betv1.2201, Bet v1.2301, Bet v1.2401, Bet v 1.2501, Bet v1.2601, Betv1.2701, Bet v1.2801, Bet v1.2901, Bet v1.3001, Bet v1.3101, Bet v6.0101, Bet v6.0102), Carpinus betulus (Car b1.0101, Car b1.0102, Carb1.0103, Car b1.0104, Car b1.0105, Car b1.0106, Car b1.0106, Carb1.0106, Car b1.0106, Car b1.0107, Car b1.0107, Car b1.0108, Carb1.0201, Car b1.0301, Car b1.0302), Corylus avellana (Cor a1.0101, Cora1.0102, Cor a1.0103, Cor a1.0104, Cor a1.0201, Cor a1.0301, Cora1.0401, Cor a1.0402, Cor a1.0403, Cor a1.0404), Ligustrum vulgare (Syrv1.0101, Syr v1.0102, Syr v1.0103), Cryptomeria japonica (Cry j2.0101,Cry j2.0102), and Cupressus sempervirens (Cup s1.0101, Cup s1.0102, Cups1.0103, Cup s1.0104, Cup s1.0105); and any variants thereof.

Lupin is an herbaceous plant of the leguminous family belonging to thegenus Lupinus. In Europe, lupin flour and seeds are widely used inbread, cookies, pastry, pasta, sauces, as well as in beverages as asubstitute for milk or soy, and in gluten-free foods. The InternationalUnion of Immunological Societies (IUIS) allergen nomenclaturesubcommittee recently designated β-conglutin as the Lup an 1 allergen.(Nadal, et al., (2012) DNA Aptamers against the Lup an 1 Food Allergen.PLoS ONE 7(4): e35253), and more recently, a high-affinity 11-mer DNAaptamer against Lup an 1 (β-conglutin) was reported (Nadal, et al.,(2013) Probing high-affinity 11-mer DNA aptamer against Lup an 1(β-conglutin). Anal. Bioanal. Chem. 405:9343-9349).

Examples of allergenic proteins from mites that can be detected usingthe compositions of the present invention include, but are not limitedto, mite (Blo t 1, Blo t 3, Blo t 4, Blot 5, Blot 6, Blot 10, Blot 11,Blot 12, Blot 13, Blot 19, Blot t 21); American house dust mite (Der f1, Der f 2, Der f 3, Der f 7, Der f 10, Der f 11, Der f 13, Der f 14,Der f 15, Der f 16, Der f 17, Der f 18, Der f 22, Der f 24);Dermatophagoides microceras (house dust mite) (Der m 1); European housedust mite (Der p 1, Der p 2, Der p 3, Der p 4, Der p 5, Der p 6, Der p7, Der p 8, Der p 9, Der p 10, Der p 11, Der p 14, Der p 15, Der p 20,Der p 21, Der p 23); Euroglyphus maynei (House dust mite) (Eur m 2, Eurm 2, Eur m 3, Eur m 4, Eur m 14); storage mite (Aca s 13, Gly d 2, Lep d2, Lep d 5, Lep d 7, Lep d 10, Lep d 13, Tyr p 2, Tyr p 3, Tyr p 10, Tyrp 13, Tyr p 24), Dermatophagoides farinae (Der f1.0101, Der f1.0102, Derf1.0103, Der f1.0104, Der f1.0105, Der f2.0101, Der f2.0102, Derf2.0103, Der f2.0104, Der f2.0105, Der f2.0106, Der f2.0107, Derf2.0108, Der f2.0109, Der f2.0110, Der f2.0111, Der f2.0112, Derf2.0113, Der f2.0114, Der f2.0115, Der f2.0116, Der f2.0117),Dermatophagoides pteronyssinus (Der p1.0101, Der p1.0102, Der p1.0103,Der p1.0104, Der p1.0105, Der p1.0106, Der p1.0107, Der p1.0108, Derp1.0109, Der p1.0110, Der p1.0111, Der p1.0112, Der p1.0113, Derp1.0114, Der p1.0115, Der p1.0116, Der p1.0117, Der p1.0118, Derp1.0119, Der p1.0120, Der p1.0121, Der p1.0122, Der p1.0123, Derp2.0101, Der p2.0102, Der p2.0103, Der p2.0104, Der p2.0105, Derp2.0106, Der p2.0107, Der p2.0108, Der p2.0109, Der p2.0110, Derp2.0111, Der p2.0112, Der p2.0113), Euroglyphus maynei (Eur m2.0101, Eurm2.0102), Lepidoglyphus destructor (Lep d2.0101, Lep d2.0101, Lepd2.0101, Lep d2.0102, Lep d2.0201, Lep d2.020) and Glycyphagusdomesticus (Gly d2.0101, Gly d2.0201); and any variants thereof.

Examples of allergenic proteins from animals that can be detected usingthe compositions of the present invention include, but are not limitedto, domestic cattle (Bos d 2, Bos d 3, Bos d 4, Bos d 5, Bos d 6, Bos d7, Bos d 8), dog (Can f 1, Can f 2, Can f 3, Can f 4, Can f 5, Can f 6),domestic horse (Equ c 1, Equ c 2, Equ c 3, Equ c 4, Equ c 5), cat (Fel d1, Fel d 2, Fel d 3, Fel d 4, Fel d 5w, Fel d 6w, Fel d 7, Fel d 8),mouse (Mus m 1), guinea pig (Cav p 1, Cav p 2, Cav p 3, Cav p 4, Cav p6), rabbit (Ory c 1, Ory c 3, Ory c 4) rat (Rat n 1), Bos domesticus(Bos d 2.0101, Bos d 2.0102, Bos d 2.0103) and Equus caballus (Equc2.0101, Equ c 2.0102); and any variants thereof.

Examples of allergenic proteins from insects that can be detected usingthe compositions of the present invention include, but are not limitedto, yellow fever mosquito (Aed a 1, Aed a 2, Aed a 3), Eastern hive bee(Api c 1), giant honeybee (Api d 1), honey bee (Api m 1, Api m 2, Api m3, Api m 4, Api m 5, Api m 6, Api m 7, Api m 8, Api m 9, Api m 10, Api m11, Api m 12), pigeon tick (Arg r 1), German cockroach (Bla g 1, Bla g2, Bla g 3, Bla g 4, Bla g 5, Bla g 6, Bla g 7, Bla g 8, Bla g 11),bumble bee (Bom p 1, Bom p 4, Bom t 1, Bom t 4), silk moth (Bomb m 1),midge (Chi k 10, Chi t 1, Chi t 1.01, Chi t 2, Chi t 2. 0101, Chi t 2.0102, Chi t 3, Chit 4, Chit 5, Chit 6, Chit 6. 01, Chit 7, Chit 8, Chit9), cat flea (Cte f 1, Cte f 2, Cte f 3), yellow hornet (Dol a 5), whiteface hornet (Dol m 1, Dol m 2, Dol m 5), biting midge (Fort 1, Fort 2),Savannah Tsetse fly (Glo m 5), Asian ladybeetle (Har a 1, Har a 2),silverfish (Lep s 1), booklouse (Lip b 1), Australian jumper ant (Myr p1, Myr p 2, Myr p 3), American cockroach (Per a 1, Per a 3, Per a 6, Pera 7, Per a 9, Per a 10), Indian meal moth (Plo i 1, Plo i 2), wasp (Pola 1, Pol a 2, Pol a 5, Pole 1, Pole 4, Pole 5, Pol f 5, Pol g 1, Pol g5, Pol m 5, Polyp 1, Polys 5, Ves vi 5), Mediterranean paper wasp (Pol d1, Pol d 4, Pol d 5), tropical fire ant (Sol g 2, Sol g 3, Sol g 4),Solenopsis invicta (red imported fire ant) (Sol I 1, Sol I 2, Sol I 3,Sol I 4), black fire ant (Sol r 2, Sol r 3), Brazilian fire ant (Sol s2, Sol s 3), horsefly (Tab y 1, Tab y 2, Tab y 5), pine processionarymoth (Tha p 1, Tha p 2), California kissing bug (Tria p 1), Europeanhornet (Vesp c 1, Vesp c 5), Vespa magnifica (hornet) (Vesp ma 2, Vespma 5), Vespa mandarinia (Giant asian hornet) (Vesp m1, Vesp m 5), yellowjacket (Ves f 5, Ves g 5, Ves m 1, Ves m 2, Ves m 5), Vespula germanica(yellow jacket) (Ves p 5), Vespula squamosa (Yellow jacket) (Ves s 1, Ves s5), Vespula vulgaris (Yellow jacket) (Ves v 1, Ves v 2, Ves v 3, Vesv 4, Ves v 5, Ves v 6), Blattella germanica (Bla g 1.0101, Bla g 1.0102,Bla g 1.0103, Bla g 1.02, Bla g 6.0101, Bla g 6.0201, Bla g 6.0301),Periplaneta Americana (Per a1.0101, Per a1.0102, Per a1.0103, Pera1.0104, Per a1.02, Per a3.01, Per a3.0201, Per a3.0202, Per a3.0203,Per a7.0101, Per a7.0102), Vespa crabo (Ves pc 5.0101, Ves pc 5.0101),Vespa mandarina (Vesp m 1.01, Vesp m 1.02); and any variants thereof.

Examples of allergenic proteins from fungi/mold that can be detectedusing the signaling polynucleotides and assays of the present inventioninclude, but are not limited to, Alternaria alternata (Alternaria rotfungus) (Alt a 1, Alt a 3, Alt a 4, Alt a 5, Alt a 6, Alt a 7, Alt a 8,Alt a 10, Alt a 12, Alt a 13), Aspergillus flavus (fungus) (Asp fl 13),Aspergillus fumigatus (fungus) (Asp f 1, Asp f 2, Asp f 3, Asp f 4, Aspf 5, Asp f 6, Asp f 7, Asp f 8, Asp f 9, Asp f 10, Asp f 11, Asp f 12,Asp f 13, Asp f 15, Asp f 16, Asp f 17, Asp f 18, Asp f 22, Asp f 23,Asp f 27, Asp f 28, Asp f 29, Asp f 34), Aspergillus niger (Asp n 14,Asp n 18, Asp n 25), Aspergillus oryzae (Asp o 13, Asp o 21),Aspergillus versicolor (Asp v 13), Candida albicans (Yeast) (Cand a 1,Cand a 3), Candida boidinii (Yeast) (Cand b 2), Cladosporiumcladosporioides (Cla c 9, Cla c 14), Cladosporium herbarum (Cla h 2, Clah 5, Cla h 6, Cla h 7, Cla h 8, Cla h 9, Cla h 10, Cla h 12), Curvularialunata (Synonym: Cochliobolus lunatus) (Cur l 1, Cur l 2, Cur l 3, Cur I4), Epicoccum purpurascens (Soil fungus) (Epi p 1), Fusarium culmorum(N.A.) (Fus c 1, Fus c 2), Fusarium proliferatum (Fus p 4), Penicilliumbrevicompactum (Pen b 13, Pen b 26), Penicillium chrysogenum (Pen ch 13,Pen ch 18, Pen ch 20, Pen ch 31, Pen ch 33, Pen ch 35), Penicilliumcitrinum (Pen c 3, Pen c 13, Pen c 19, Pen c 22, Pen c 24, Pen c 30, Penc 32), Penicillium crustosum (Pen cr 26), Penicillium oxalicum (Pen o18), Stachybotrys chartarum (Sta c 3), Trichophyton rubrum (Tri r 2, Trir 4), Trichophyton tonsurans (Tri t 1, Tri t 4), Psilocybe cubensis (Psic 1, Psi c 2), Shaggy cap (Cop c 1, Cop c 2, Cop c 3, Cop c 5, Cop c 7),Rhodotorula mucilaginosa (Rho m 1, Rho m 2), Malassezia furfur (Malaf2,Malaf3, Malaf4), Malassezia sympodialis (Malas1, Malas5, Malas6, Malas7,Malas8, Malas9, Malas10, Malas1l, Malas12, Malas13) and Alternariaalternate (Alt a1.0101, Alt a1.0102); and any variants thereof.

Examples of additional allergens include, but are not limited to,Nematode (Ani s 1, Ani s 2, Ani s 3, Ani s 4), worm (Asc s 1), softcoral (Den n 1), rubber (Latex) (Hev b 1,hev b 2,hev b 3,hev b 5,hev b6,hev b 7,hev b 8,hev b 9,hev b 10,hev b 11,hev b 12,hev b 13), obeche(Trip s 1) and Heveabrasiliensis (Hey b6.01,hev b6.0201,hev b6.0202,hevb6.03, Hey b8.0101,hev b8.0102,hev b8.0201,hev b8.0202,hev b8.0203,hevb8.0204,hev b10.0101,hev b10.0102,hev b10.0103,hev b11.0101,hevb11.0102); and any variants thereof.

In some embodiments, SPNs and compositions of the present invention maybe used in a hospital for clinical food allergy or allergy test and toidentify food/allergen(s) to which a patient is allergic. In addition,SPNs and compositions of the present invention may be used as a carry-ontester for people who have food/environmental allergy, for example athome to test commercial food, or at restaurant to check dishes theyordered. The food sample could be fresh food, frozen food, cooled foodor processed food containing animal derived meat and/or vegetables.

Other Target Molecules

In some embodiments, SPNs and compositions of the present invention maydetect other target molecules, including but not limited to, pathogensfrom a pathogenic microorganism in a sample, such as bacteria, yeasts,fungi, spores, viruses or prions; disease proteins (e.g., biomarkers fordiseases diagnosis and prognosis); pesticides and fertilizers remainedin the environment; and toxins. In other embodiments, SPNs andcompositions of the present invention may bind to non-protein targetssuch as minerals and small molecules (e.g., antibiotics).

Applications

In accordance with the present invention, detection molecules, signalingpolynucleotides (SPNs), compounds and compositions of the presentinvention may be used to, in a broad concept, detect any proteins in asample in a large variety of applications, such as food safety,diagnostic and prognostic tests in civilian and battlefield settings,environmental monitoring/control, and military use for detection ofbiological weapons. In even broader applications, the detectionmolecules, signaling polynucleotides (SPNs), compounds and compositionsof the present invention may be used to detect any substances to whichnucleic acid-based detection molecules bind, such as minerals in water.

The applications in food safety control may include, but are not limitedto, detecting and monitoring food contaminants (e.g., pathogens andtoxins), food quality (e.g., nutrients), diet supplements, and foodallergens. The applications in a battle field setting may include, butare limited to, testing of antibiotics and biological drugs, biologicalweapons, infectious diseases monitoring and food safety.

Various methods and assays may be used in combination with the detectionmolecules, signaling polynucleotides, compounds and compositions of thepresent invention; the choice may depend on the application field.

Detection Methods and Assays: Food Allergens

In some embodiments, analytical assays and methods for detecting variousallergens (e.g., food allergens) in samples are provided. Assays andmethods provided can detect the presence or absence of an allergen ofinterest in a sample, and/or determine the amount of the allergen in asample.

In some embodiments, methods for detecting one or more allergens in atest sample, such as a food sample, comprise the steps (a) obtaining atest sample suspected of containing an allergen; (b) processing the testsample and extracting proteins from the processed sample using anextraction buffer; (c) mixing the protein extraction of step (b) with aSPN that specifically binds to the allergen; (d) activating the sampleand SPN mixture by means of an energy excitation; and (e) visualizingthe interaction between the SPN and the allergen protein and detectingthe absence or presence of the allergen in the test sample. In someembodiments, a light-emitting diode (LED) light may be used as anexcitation means.

Sample Processing and Extraction Buffer

The ability of a detection assay and method to detect allergen proteinsin a test sample is affected by the efficiency with which these proteinsare extracted from the samples, in addition to the efficiency with whichthe detection molecules used in the present invention to detect theseproteins in the sample extract. In some embodiments, samples areprocessed and allergen proteins are extracted to ensure a fast, reliableand sensitive detection assay. The sample size and weight, extractionsolution and extraction process may be optimized for an effective andnon-destructive reaction. Any mechanisms that can break samples such ascutting, grinding, homogenization and filtration may be used, alone orin combination, to process a sample.

In some embodiments, a universal protein extraction buffer may be usedto retrieve enough target proteins (e.g. allergens) (minimum 2 mg/mltotal protein) for analysis from any food matrix. In some embodiments,the formulation of the universal protein extraction buffer can extractthe protein at room temperature and in minimal time. In some aspects,allergen proteins may be extracted in less than about 2 minutes, or lessthan about 1 minute, or less than about 30 seconds. The buffer may needto be incorporated with an extraction protocol that will include foodsampling, homogenization and filtration. The extraction protocol may beimplemented in a way that is efficient and repeatable over time and indifferent food matrices. This universal formulation will be clinicallyrelevant as to try to minimally effect the food tested and only sampleapproximately 0.5 g of food, allowing to detect traces of allergenstheir concentration will be minimal in the sample. This optimizedprotein extraction process will provide a fast, accurate and universalprotocol that allows detection of an allergen in any food matrix.

This universal extraction buffer can maximize protein extraction andallergen retrieval. The universal extraction buffer will be applicableto any allergen and to all foods (e.g. pre-processed or post-processed).Additionally, the universal extraction buffer can improve signalingpolynucleotides (SPNs) binding affinity, minimize non-specific bindingand increase signal to noise ratio.

Allergen Detection Assays

In some embodiments, compositions, compounds and signalingpolynucleotides of the present invention may be used to replaceantibodies as an alternative molecular recognition element inenzyme-linked immunosorbent assay (ELISA). The application of aptamerbased signaling polynucleotides in ELISA gives rise to an ELISA-derivedassay called enzyme-linked apta-sorbent assay (ELASA). As with the ELISAmethod, ELASA can be used in several different configurations, includingdirect, indirect, and sandwich assays (Toh et al., Biosens. Bioelectron,2015, 64, 392-403, the content of which is incorporated herein byreference in its entirety.)

In some embodiments, compositions, compounds and signalingpolynucleotides of the present invention may be used in a real-timeapta-PCR for detection of a target protein in a sample. In this assay,the target in the test sample and immobilized same target will competefor aptamer binding. Following competition, any aptamer bound to theimmobilized target protein can be heat-eluted and quantitativelyamplified using real-time PCR. Aptamers used for this assay can belabel-free (Pinto et al., Anal-Bioanal Chem., 2014, 406(2), 515-524; andSvobodova et al., Food Chem., 2014, 165, 419-423; the content of each ofwhich is incorporated herein in its entirety.)

In some embodiments, allergen detection assays may depend onfluorescence emission signal from fluorescence resonance energy transfer(FRET). Signaling polynucleotides (SPNs) is labeled with fluorophore atthe ends of the sequences. The specific interaction with a targetinduces a change in the bi-aptameric structure resulting in an increasein fluorescence emission. The method is highly specific and sensitive.

In certain embodiments, one or more signaling polynucleotides (SPNs) maybe used, depending on the nature of the food matrixes. Some foodcontains several allergenic proteins, e.g., at least eight peanutproteins, such as Ara h1 and Ara h2, can potentially cause animmunological response. In such case, more than one signalingpolynucleotides (SPNs) against more than one allergenic protein may beused in a mixed cocktail for detecting the absence or presence ofpeanut. In other aspects, some food matrixes such as fish, shellfish andmollusks, contain only one major allergenic protein. One or more SPNsthat specifically bind to this major allergen protein may be used forallergen detection.

In order to provide an accurate and reliable detection result in anallergen detection assay, total proteins extracted from a test sampleare measured. The total proteins extracted from a test sample may bedetermined using any protein assays known to a skilled artisan in thefield, e.g., bicinchoninic acid assay (BCA). In some aspects, a proteinindication molecule (e.g., Pyrogalbl Red Molybdate, PRM) is used todetermine the total protein. Any signal detected from the detectionmolecule-allergen interaction will be nominated by the total proteinmeasurement.

In some embodiments, allergen detection assays and methods of thepresent invention provide a calibration standard (i.e. calibrationcurves) for a particular allergen and a SPN used. The calibrationstandard of a particular allergen protein may be generated from a raw orprocessed material that contains such allergen, or a purified allergen.

In some embodiments, allergen detection assays and methods of thepresent invention can detect a lower concentration of allergen in a foodsample. The sensitivity of nucleic acid aptamers makes it possible todetect the presence of an allergen as low as 0.0001 ppm. In someaspects, the concentration or mass of allergen that can be detected mayrange from 0.001 ppm to 5 ppm, or from 0.001 ppm to 0.1 ppm, or from 0.1ppm to 3 ppm, or from 1 ppm to 5 ppm, or from 5 ppm to 10 ppm. In someaspects, the concentration or mass of allergen in a food sample that canbe detected may be 0.001 ppm, 0.002 ppm, 0.003 ppm, 0.004 ppm, 0.005ppm, 0.006 ppm, 0.007 ppm, 0.008 ppm, 0.009 ppm, 0.01 ppm, 0.02 ppm,0.03 ppm, 0.04 ppm, 0.05 ppm, 0.06 ppm, 0.07 ppm, 0.08 ppm, 0.09 ppm,0.1 ppm, 0.2 ppm, 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6 ppm, 0.7 ppm, 0.8 ppm,0.9 ppm, 1.0 ppm, 1.5 ppm, 2 ppm, 2.5 ppm, 3 ppm, 3.5 ppm, 4 ppm, 4.5ppm, 5 ppm or 10 ppm.

In some embodiments, allergen detection assays and methods of thepresent invention may complete the implementation in less than 5minutes. In some aspects, the assay time may be from about 1 minute toabout 5 minutes, about 1 minute to about 3 minute, about 2 minutes toabout 10 minutes, about 5 minutes to about 10 minutes. In other aspects,the assay time may last less than 1 min, 2 min 3 min, 4 min, 5 min, 6min, 7 min, 8 min, 9 min, or 10 min. In further other aspects, the assaytime may last less than about 10 seconds, about 15 seconds, about 20seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40seconds, about 45 seconds, about 50 seconds, about 55 seconds or about60 seconds.

Detection System and Display Platform

Methods and systems used to detect and display aptamer and proteininteraction may be used to display the detection results.

A commonly used method in the field is the use of electrochemicalindicators which detect mass and charge transfer during aptamer andtarget interaction. According to this method, Aptamers are loaded to anelectrode and an electrochemical indicator is bound to a target ofinterest. Electrochemical indicators may include, but are not limitedto, methylene blue (MB).

Some non-limiting examples of methods for detection of aptamer-targetinteraction include an assay for the direct detection of cancer cellsusing aptamer-conjugated gold nanoparticles (ACGNPs) selective for cellsurface molecules on CCRF-CEM cells (CCL-119 T-cell, human acutelymphoblastic leukemia) and Ramos cells (CRL-1596, B-cell, humanBurkitt's lymphoma) (Medley, et al., Gold Nanoparticle-BasedColorimetric Assay for the Direct Detection of Cancerous Cells. Anal.Chem. 2008, 80:1067-1072); the use of aptamer-linked gold nanoparticles(AuNPs) that undergo fast disassembly into red dispersed nanoparticlesupon binding of target analytes (Lu, et al. Chapter 14:Nanoparticles/Dip Stick, in Nucleic Acid and Peptide Aptamers: Methodsand Protocols, Günter Mayer (ed.). 535:223-239); and a differentialpulse voltammetry (DPV)-based biosensor employing aptamer-AuNPconjugates as the sandwich-amplification element for the ultrasensitivedetection of IgE in human serum (over a range 1-10,000 ng/mL with an LODas low as 0.52 ng/mL) (Wang, et al., Aptamer-Au NPsconjugates-accumulated methylene blue for the sensitive electrochemicalimmunoassay of protein, Talanta, 15 Apr. 2010, 81(1-2):63-67).

However, at least one disadvantage shared by many electrochemicalbiosensors is the off-line nature of the measurements, requiring longincubation times with analyte solution, rather than real-time detection(Pilloli, et al., Advances in biosensor development based on integratingnanotechnology and applied to food-allergen management. Trends inAnalytical Chemistry, June 2013, 47:12-26).

In accordance with the present invention, an optical assembly may beused to detect the interaction between a SPN and a target allergen. Theoptical assembly may comprise a light emitting diode (LED) that provideslight of an excitation wavelength appropriate to excite the fluorophoreof the signaling polynucleotides. The fluorescence emitted from thefluorophores of the SPNs may be filtered and only the wavelength(s) ofinterest is transmitted. A means then may be used to process and convertthe fluorescence signals to useful readouts (i.e. digital signals).

The detection result from the present assay may be displayed in aplatform that a user can easily read such as a display window. In oneembodiment, it may be a platform application in a cellphone (Coskun etal., A personalized food allergen testing platform on a cellphone, LabChip., 2013, 13(4), 636-640; the content of which is incorporated hereinby reference in its entirety.)

Formulations, Packaging, Kits, Devices and Systems

Formulations: Detection molecules, compounds, signaling polynucleotidesof the present invention may be formulated following standardprocedures. In some embodiments, detection molecules, SPNs of thepresent invention may be formulated in a solution which favors theinteraction between the detection molecules and the allergen.

Packaging: Formulations and/or compositions of detection molecules,signaling polynucleotides of the present invention can be packaged foruse in a variety of pharmaceutically or diagnostically acceptablecontainers using any acceptable container closure, as the formulationsare compatible with PVC-containing and PVC-free containers and containerclosures. Examples of acceptable containers include, but are not limitedto, ampules and pre-filled syringes, cartridges and the like.

Alternatively, the formulation may contain lyophilized aptamer in onecompartment of an admix bag and an acceptable solvent in a separatecompartment of the admix bag such that the two compartments may be mixedtogether prior to administration to a patient. Acceptable containers arewell known in the art and commercially available. Preferably, theformulations are stored in a Type 1 glass vial with a butyl rubberstopper. The formulations in liquid form may be stored in a refrigeratedenvironment. Alternatively, the lyophilized formulations may be storedat room temperature, or refrigerated or frozen.

Preferably, the formulations are sterile. A “sterile” formulation, asused herein, means a formulation that has been brought to a state ofsterility and has not been subsequently exposed to microbiologicalcontamination, i.e., the container holding the sterile composition hasnot been compromised. Sterile compositions are generally prepared bypharmaceutical manufacturers in accordance with current GoodManufacturing Practice (“cGMP”) regulations of the U.S. Food and DrugAdministration.

In some embodiments, sterile pharmaceutical formulations can be preparedusing aseptic processing techniques. Sterility is maintained by usingsterile materials and a controlled working environment. All containersand apparatus are sterilized, preferably by heat sterilization, prior tofilling. Then, the container is filled under aseptic conditions, such asby passing the composition through a filter and filling the units.Therefore, the formulations can be sterile filled into a container toavoid the heat stress of terminal sterilization.

In some embodiments, the formulations are terminally sterilized usingmoist heat. Terminal sterilization can be used to destroy all viablemicroorganisms within the final, sealed container containing thepharmaceutical formulation. An autoclave is typically used to accomplishterminal heat-sterilization of drug products in their final packaging.Typical autoclave cycles in the pharmaceutical industry to achieveterminal sterilization of the final product are 121° C. for at least 10minutes.

Kits: detection molecules, compounds and compositions of the presentinvention may be combined with other ingredients or reagents or preparedas components of kits or other retail products for commercial sale ordistribution. The kit will contain the compound or composition, alongwith instructions regarding administration and/or use of the kit. Thekit may also contain one or more of the following: a syringe, a bag orbottle.

Devices and Systems: The signaling polynucleotides, compounds andcompositions of the present invention may be used in any allergendetection devices and systems. Some non-limiting examples includelateral flow devices (LFD), microfluidic chips (U.S. Pat. No.8,617,903), portable detection devices/systems described in the commonlyowned U.S. patent application No. 62/133,632 filed on Mar. 16, 2015 andthe cartridge as described in the commonly owned PCT patent applicationNO.: PCT/US14/62656 filed on Oct. 28, 2014, each of which isincorporated herein by reference in its entirety.

Definitions

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual sub-combination of the members of such groupsand ranges. The following is a non-limiting list of term definitions.

About: As used herein, the term “about” when referring to a measurablevalue such as an amount of weight, time, dose, etc. is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

Activity: As used herein, the term “activity” refers to the condition inwhich things are happening or being done. Compositions of the inventionmay have activity and this activity may involve the binding to a targetmolecule.

Allergen: as used herein, the term “allergen” means a compound,substance or composition that causes, elicits or triggers and immunereaction in a subject. As such, allergens are typically referred to asantigens. An allergen is typically a protein or a polypeptide.

Allergen detection molecule: As used herein, the term “an allergendetection molecule” refers to Any molecule which is capable of, or does,interact with and/or bind to one or more allergens in a way that allowsdetection of such allergen in a sample is referred to herein as an“allergen detection molecule” or “detection molecule”.

Binding affinity: As used herein, the term “binding affinity” refers tothe tendency of a detection molecule (e.g., aptamer) to bind or not binda target (e.g., allergen) and describes the measure of the strength ofthe binding or affinity of the detection molecule to bind the target.

Biomolecule: As used herein, the term “biomolecule” is any naturalmolecule which is amino acid-based, nucleic acid-based,carbohydrate-based or lipid-based, and the like.

Complementary and substantially complementary: As used herein, the term“complementary” refers to the ability of polynucleotides to form basepairs with one another. Base pairs are typically formed by hydrogenbonds between nucleotide units in antiparallel polynucleotide strands.Complementary polynucleotide strands can form base pair in theWatson-Crick manner (e.g., A to T, A to U, C to G), or in any othermanner that allows for the formation of duplexes. As persons skilled inthe art are aware, when using RNA as opposed to DNA, uracil rather thanthymine is the base that is considered to be complementary to adenosine.However, when a U is denoted in the context of the present invention,the ability to substitute a T is implied, unless otherwise stated.Perfect complementarity or 100% complementarity refers to the situationin which each nucleotide unit of one polynucleotide strand can formhydrogen bond with a nucleotide unit of a second polynucleotide strand.Less than perfect complementarity refers to the situation in which some,but not all, nucleotide units of two strands can form hydrogen bond witheach other. For example, for two 20-mers, if only two base pairs on eachstrand can form hydrogen bond with each other, the polynucleotidestrands exhibit 10% complementarity. In the same example, if 18 basepairs on each strand can form hydrogen bonds with each other, thepolynucleotide strands exhibit 90% complementarity.

Detection: As used herein, the term “detection” means an extraction of aparticular target protein from a mixture of many non-target proteins,indicating the absence, presence, and/or amount of a target protein froma mixture of many non-target proteins.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties which are attached, incorporated orassociated with another entity, which markers, signals or moieties arereadily detected by methods known in the art including radiography,fluorescence, chemiluminescence, enzymatic activity, absorbance,immunological detection and the like. Detectable labels may includeradioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions,ligands, biotin, avidin, streptavidin and haptens, quantum dots,polyhistidine tags, myc tags, flag tags, human influenza hemagglutinin(HA) tags and the like. Detectable labels may be located at any positionin the entity with which they are attached, incorporated or associated.For example, when attached, incorporated in or associated with a peptideor protein, they may be within the amino acids, the peptides, orproteins, or located at the N- or C-termini.

Including: As used herein, the term “including” refers to” including butnot limited to”. “Including” and “including but not limited to” are usedinterchangeably.

Interaction: As used herein, the term “interaction” refers to a kind ofaction that occurs as two or more molecules have effect upon oneanother. In the context of the present invention, an interaction betweena detection molecule and a target affects the structure of the detectionmolecule and such effect will generate energetic changes that can bevisualized.

Pathogen: As used herein, the term “pathogen” means anydisease-producing agent (especially a virus or bacterium or othermicroorganism).

Polynucleotide: As used herein, the term “polynucleotide” refers tonucleobase polymers or oligomers in which the nucleobases are connectedby sugar phosphate linkages (sugar-phosphate backbone). Exemplary poly-and oligonucleotides include polymers of 2′ deoxyribonucleotides (DNA)and polymers of ribonucleotides (RNA). A polynucleotide may be composedentirely of ribonucleotides, entirely of 2′ deoxyribonucleotides orcombinations thereof.

Polynucleotide variants: As used herein, the term “polynucleotidevariants” refers to molecules with some differences in their nucleicacid sequences as compared to a native or starting sequence.

ppm: As used herein, the term “ppm” is an abbreviation of parts permillion. ppm is a value that represents the part of a whole number inunits of 1/1000000. ppm is dimensionless quantity, a ratio of 2quantities of the same unit. For example: mg/kg. One ppm is equal to1/1000000 of the whole: 1 ppm= 1/1000000=0.000001=1×10⁻⁶. ppm herein isused to measure chemical (protein) concentration, usually in a solution.Solute concentration of 1 ppm is solute concentration of 1/1000000 ofthe solution. The concentration C in ppm is calculated from the solutemass m_(solute) in milligrams and the solution mass m_(solution) inmilligrams: C_((ppm))=1000000×m_(solute)/(m_(solution)+m_(solute))

Sample: As used herein, the term “sample” refers to any composition thatmight contain a target of interest to be analyzed including, but notlimited to, biological samples obtained from subjects (including humansand animals as detailed below), samples obtained from the environmentfor example soil samples, water samples, agriculture samples (includingplant and crop samples), or food samples. Food samples may be obtainedfrom fresh food, processed/cooked food or frozen food.

Sensitivity: As used herein, the term “sensitivity” means the ability ofa detection molecule to bind to a target molecule.

Specifically bind(s): As used herein, the term: specifically bind(s)”means that a detection molecule (e.g., aptamer) reacts or associatesmore frequently, more rapidly, with greater duration and/or with greateraffinity with a particular target such as an allergen protein than itdoes with alternative targets. For example, an aptamer that specificallybinds to an allergen protein binds that protein or a fragment thereofwith greater affinity, avidity, more readily, and/or with greaterduration than it binds to unrelated protein and/or the fragmentsthereof. It is also understood by an artisan by this definition, forexample, a detection molecule (e.g., aptamer) that specifically binds toa first target may or may not specifically bind to a second target. Assuch, “specific binding” does not necessarily require exclusive bindingor non-detectable binding of another molecule, this is encompassed bythe term “selective binding”. Generally, but not necessarily, referenceto binding means specific binding. The specificity of binding is definedin terms of the comparative dissociation constants (Kd) of the aptamerfor target as compared to the dissociation constant with respect to theaptamer and other materials in the environment or unrelated molecules ingeneral. Typically, the Kd for the aptamer with respect to the targetwill be 2-fold, 5-fold, or 10-fold less than the Kd with respect to thetarget and the unrelated material or accompanying material in theenvironment. Even more preferably, the Kd will be 25-fold, 50-fold,75-fold, 100-fold, 150 fold or 200-fold less.

Target: as used herein, the term “target” and “target molecule” refersto a molecule which may be found in a tested sample and which is capableof binding to a detection molecule such as an aptamer or an antibody.

Universal buffer: As used herein, the term “universal buffer” refers toa buffer that may be used for a variety of samples.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or the entiregroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anyantibiotic, therapeutic or active ingredient; any method of production;any method of use; etc.) can be excluded from any one or more claims,for any reason, whether or not related to the existence of prior art.

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

EXAMPLES Example 1 Design of Aptamers as Signaling Polynucleotides

In this proof-of-concept example, two previously known aptamer sequenceswere used to design three different signaling polynucleotides. Anaptamer against the Ara h 1 protein allergen is described by Tran et al.in Selection of aptamers against Ara h 1 protein for FO-SPR biosensingof peanut allergens in food matrices. Biosensors and Bioelectronics,2013, 43, 245-251 (incorporated herein by reference in entirety). Thesequence of this aptamer is shown below.

(SEQ ID NO: 1) 5′CGCACATTCCGCTTCTACCGGGGGGGTCGAGCTGAGTGGATGCGAATCTGTGGGTGGGCCGTAAGTCCGTGTGTGCGAA3′

The original aptamer of SEQ ID NO: 1 was modified to add a 5′-T residueto improve the functioning of the fluorophore-quencher pair. Fluoresceinwas then linked to the 5′-T residue as shown below.

(SEQ ID NO: 2) 5′FluoresceinTCGCACATTCCGCTTCTACCGGGGGGGTCGAGCTGAGTGGATGCGAATCTGTGGGTGGGCCGTAAGTCCGTGTGTGCGAA3′

A 9-nucleotide linker with a 3′-DABCYL quencher was designed as shownbelow to be complementary to the first ten residues of the 5′-end of theT-modified aptamer of SEQ ID NO: 2.

(SEQ ID NO: 3) 3′DABCYLAGCGTGTAA5′

The 9-nucleotide linker (SEQ ID NO: 3) was then annealed to the 5′-endof the main modified anti-peanut allergen aptamer sequence (SEQ ID NO:2) to bring the fluorescein fluorophore into proximity with the DABCYLquencher moiety. The structure of the assembled signaling polynucleotidefor detection of peanut allergen Ara h 1 is shown below.

     3′DABCYLAGCGTGTAA5′ (SEQ ID NO: 3)              |||||||||5′FluoresceinTCGCACATTCCGCTTCTACCGGGGGGGTCGAGCTGAGTGGATGCGAATCTGTGGGTGGGCCGTAAGTCCGTGTGTGCGAA3′ (SEQ ID NO: 2)

The signaling polynucleotide prepared from annealing SEQ ID NOs: 2 and 3is a dimeric entity herein designated SPN-A*. The Secondary structure ofSPN-A* is shown in FIG. 1. The arrangement of the components of thesignaling polynucleotide 200 (SEQ ID NO. 2) are core sequence 202 (SEQID NO. 1), fluorophore 404, quencher 406 and liner sequence 208 (SEQ IDNO. 3).

In a similar manner, a signaling polynucleotide was designed based uponthe sequence of an aptamer against egg white lysozyme described by Tranet al. in Selection and Characterization of DNA Aptamers for Egg WhiteLysozyme. Molecules 2010, 15(3), 1127-1140 (incorporated herein byreference in entirety). The sequence of this aptamer is shown below.

(SEQ ID NO: 4) 5′GCAGCTAAGCAGGCGGCTCACAAAACCATTCGCATGCGGC3′

The original aptamer of SEQ ID NO: 4 was modified to add a 5′-T residueto improve the functioning of the fluorophore-quencher pair. Fluoresceinwas then linked to the 5′-T residue as shown below.

(SEQ ID NO: 5) 5′FluoresceinTGCAGCTAAGCAGGCGGCTCACAAAACCATTCGCATG CGGC3′

A 10-nucleotide linker with a 3′-DABCYL quencher was designed as shownbelow to be complementary to the first ten residues of the 5′-end of theT-modified aptamer of SEQ ID NO: 5.

(SEQ ID NO: 6) 3′DABCYLACGTCGATTC5′

The 10-nucleotide linker (SEQ ID NO: 6) was then annealed to the 5′-endof the main modified anti-lysozyme aptamer sequence (SEQ ID NO: 5) tobring the fluorescein fluorophore into proximity with the DABCYLquencher moiety. The structure of the assembled signaling polynucleotidefor detection of lysozyme is shown below.

      3′DABCYLACGTCGATTC5′ (SEQ ID NO: 6)               ||||||||||5′-FluoresceinTGCAGCTAAGCAGGCGGCTCACAAAACCATTCGCAT GCGGC3′(SEQ ID NO: 5)

The dimeric signaling polynucleotide prepared from SEQ ID NOs: 5 and 6is herein designated SPN-E*. A reaction between SPN-E* and lysozyme isshown schematically in FIG. 3. The arrangement of the components of thesignaling polynucleotide SPN-E* 400 (SEQ ID NO. 5) are core sequence 402(SEQ ID NO. 4), fluorophore 404, quencher 406 and liner sequence 408(SEQ ID NO. 6), thereby allowing the fluorophore 404 to fluoresce uponexcitation.

A third signaling polynucleotide was designed based upon the aptamersequence of SEQ ID NO: 4 described above. A 5′-T residue was appended toSEQ ID NO: 4 and the 3′-end was modified by addition of a fivenucleobase segment complementary to the last five nucleobases of the5′-end of the original aptamer sequence of SEQ ID NO: 4. Then5′-fluorescein and 3′-DABCYL moieties were linked to produce thesequence shown below (SEQ ID NO: 7) wherein the additional fivenucleobase segment is underlined along with the first five nucleobasesat the 5′-end of the original aptamer sequence (not including the addedthe 5′-T residue).

(SEQ ID NO: 7) 5′FluoresceinTGCAGCTAAGCAGGCGGCTCACAAAACCATTCGCATGCGGCGCTGCDABCYL3′

This signaling polynucleotide is a hairpin entity herein designatedSPN-E. It will be recognized that the underlined residues as the 5′-endand the 3′-end are complementary for the purpose of forming a hairpinsecondary structure as shown in the leftmost structure of FIG. 2 (coresequence 302 (SEQ ID NO. 4). This structure brings the fluorophore 304and quencher 306 into close proximity with each other to allow thequencher 306 to quench the fluorophore 304. The binding of the signalingpolynucleotide to lysozyme can disrupt the hybridization of the two endsof the signaling polynucleotide 300 (SEQ ID NO. 7) as shown in therightmost structure of the core sequence 302 (SEQ ID NO. 4), resultingin separation of the fluorophore 304 from the quencher 306, therebyactivating the fluorophore 304.

Example 2 Selection and Optimization of Aptamer Polynucleotides

An in vitro screening experiment based on SELEX method was carried outand aptamers were selected against the allergen targets including egg,gluten, milk, soy, fish, peanut, cashew and crustacean, over thecounter-target (combinations of the non-target proteins) and werefurther engineered for their capability in detecting targeted foodallergens.

Experimental Plan

Various RNA libraries were used to select for binding ability inselection buffer consisting of 100 mM Tris (pH 8), 5 mM EDTA, 150 mMNaCl, 10 mM MgCl2, 0.1% SDS, 0.1% Gelatin, 1% NP-40 (Tergitol), 0.5%Deoxycholate Sodium at 23° C. A given round of selection began withincubating RNA library members in either the buffer alone (negativeselection), then collecting the portion of the library that did notrespond (i.e. cleave). The second part of each round (when called for)consisted of incubating the non-responsive molecules from the priornegative selection step with the full combination of non-positivetargets (as the counter), or with just the selection buffer again for asecond negative selection. Once again, the non-responsive (non-cleaving)molecules would be collected. The final step of each round consists ofincubating the material from the previous step with the positive target(each of the allergens as appropriate) in buffer, then collecting theresponsive material (i.e. cleaved RNA). Each selection round wasfollowed by reverse transcription to generate cDNA, libraryamplification through PCR, and regeneration of the RNA library bytranscription. After subjecting the initial library of diverse randomsequences to varying consecutive rounds of selection (i.e. negative,counter and positive selections), again project-dependent, and theenriched libraries were divided into three fractions to perform theparallel assessments.

The parallel assessment of libraries enriched after rounds of negative,counter and positive selections, involves simultaneously exposing onethird of the enriched library to selection buffer alone, anotherone-third to the counter-target complex in selection buffer, and thefinal one-third of the enriched library to the target allergen inbuffer. Any residual RNA molecules that react indiscriminately to bothtarget allergen and counter-targets, or that still generate a responsein the absence of the target allergen were identified and discardedduring further bioinformatics analysis.

The enriched RNA libraries after the parallel assessment were subjectedto PAGE gel assessment. 40 pmoles of enriched library was exposedseparately to either the negative (buffer only), counter target, ortarget allergen (e.g., milk, wheat, egg white and peanut) in selectionbuffer. After 5 minutes incubation at 23° C., libraries exhibiting apositive response (i.e. cleavage) material were collected, ethanolprecipitated, reverse transcribed, and PCR-amplified for sequencing andbioinformatics analysis.

Materials and Methods

Targets (complexes of proteins from cashew, peanut, fish, milk, soy,gluten, egg and crustacean) were dried down, if necessary, before beingcombined with RNase-free water for preliminary analysis and aptamerscreening. When needed, targets were pooled to produce counter-targetmixture by combining appropriate amounts of the targets which were notdesignated as positive target for the selection. The initial aptamerlibrary template and primers were synthesized by IDT (Coralville, Iowa)as single-stranded DNA. The library was then primer extended to providedouble-stranded DNA (dsDNA) using Titanium Taq DNA polymerase fromClontech (Mountain View, Calif.).

Following the experimental plan, for a given generation of the library,RNA was transcribed from the previous dsDNA with Ampli Scribe T7Transcription kits from Epicentre (Madison, Wis.) and purified using a10% denaturing polyacrylamide gel electrophoresis (PAGE). The purifiedRNA was combined with Selection Buffer, which was then diluted to 1×concentration (100 mM Tris (pH 8), 5 mM EDTA, 150 mM NaCl, 10 mM MgCl2,0.1% SDS, 0.1% Gelatin, 1% NP-40 (Tergitol), 0.5% Deoxycholate Sodium)for negative selection. Negative selection began with a refolding cycle,which involved heating the sample to 65° C. to denature the RNA beforebringing the sample to 23° C. for the remainder of the incubation. Afterincubation, non-cleaved RNA was separated from cleaved RNA using 10%denaturing PAGE. Recovered non-cleaved material was combined withcounter-target and buffer, target and buffer, or buffer alone dependingon the selection step, incubated at 23° C., and partitioned on 10%denaturing PAGE. Recovery and another selection step was implemented ifcalled for. cDNA was then generated from eluted post-selection libraryusing SuperScript II Reverse Transcriptase (Life Technologies; Carlsbad,Calif.), then PCR-amplified with Titanium Taq DNA polymerase (Clontech;Mountain View, Calif.) to complete the round of selection. After severalrounds of selection steps, libraries were enriched and showed that thenegative cleavage amount was less than 30%, and that there was at least5% more cleavage in the positive treatment when compared to the counter.

The initial libraries consisting of approximately 10¹⁴ random sequenceswas subjected to varying rounds of ribozyme-based SELEX to enrich forsequences that bind to the target allergens and to eliminated sequencesthat bind to the counter-targets over multiple rounds of selection. As aresult, the population to be sequenced is expected to contain multiplecopies of potential aptamer candidates (Van Simaeys et al., Study of theMolecular Recognition of Aptamers Selected through Ovarian CancerCell-SELEX, 2010, PLOS One, 5(11): e13770).

Sequencing and Bioinformatics

The Illumina (San Diego, Calif.) MiSeq system was implemented tosequence the aptamers after the selections using a paired-end readtechnique. Bioinformatics analysis of the sequencing data identifiedcandidate aptamer molecules. The deep sequencing and subsequent dataanalysis reduced the traditional approach of performing a large numberof selections, which may introduce error and bias due to the screeningprocess (Schülze et al., Probing the SELEX Process with Next-GenerationSequencing, PLos One, 2011, 6(12): e29604).

Aptamers Candidate Selection

Sequence family construction focused on motif presence which means thata sequence's frequency in the positive target population was factoredin, but places greater emphasis on the prevalence of sub-sequences inthe overall population (100% match over the entire sequence notnecessary to join a family). Two other factors were used to adjust theimportance of motif-family size to determine candidate sequences. Onefactor is the presence of the sequence in the negative andcounter-target population. Three libraries were collected from theparallel assessment: the positive target-exposed library, thebuffer-only negative library, and the counter-target-exposed library.All libraries were analyzed to discover any sequences that have yet tobe removed during a negative- or counter-selection step, but still haveaffinity for both the target and counter-target. A given sequenceappears more frequently in the positive population than in thecounter-target-exposed population, making it an attractive candidate forfurther testing.

The secondary structure of a given candidate sequence was also predictedusing the Mfold secondary structure modeling software (Zucker, Mfold webserver for nucleic acid folding and hybridization prediction, NucleicAcids Res., 2003, 31 (13): 3406-3415).

A set of aptamer sequences were selected and further designed assignaling polynucleotides for detecting different food allergens,including peanut, egg white, wheat and milk. The selected aptamers arelisted in Table 1. The selected aptamers for each food allergen are thenfurther modified at either one or both of the 5′ end and the 3′end tooptimize the binding affinity to its targeted allergen. Modifiedsequences that are intended to have a fluorescein (e.g., FITC/FAMmolecule) on the 5′end and a quencher on the 3′ end are the signalingpolynucleotides that will be tested for allergen detection as describedherein.

Example 3 Total Protein Measurement

Total protein measurement is tested using Pyrogallol Red-molybdate (PRM)protein dye-binding assays. PRM is first made in a solution containing0.156 mM pyrogallol red, 0.209 mM sodium molybdate and 50 mM Tris-HCl. Atest plate is prepared by adding 20 μl/well PRM solution and the plateis dry overnight. After processing the test food matrixes, processedsample solution (400 μl) is added to each well and the proteinabsorbance is read immediately at 600 nm.

What is claimed is:
 1. A signaling polynucleotide (SPN) for detecting anallergen comprising a nucleotide sequence selected from the groupconsisting of sequences presented by SEQ ID NOs.:8-26.
 2. The SPN ofclaim 1 wherein the nucleotide sequence or a portion of the nucleotidesequence specifically binds to the allergen.
 3. The SPN of claim 2further comprising a fluorophore and a quencher which are in sufficientproximity for the quencher to quench the fluorescence of thefluorophore.
 4. The SPN of claim 3 wherein the fluorophore and thequencher are linked to opposite ends of the sequence of the SPN, andwherein 5 to 20 nucleobase residues at the 5′-end of the sequence are atleast 80% complementary to 5 to 20 nucleobase residues at the 3′-end ofthe sequence capable of forming a hairpin structure, thereby bringingthe quencher in sufficient proximity to the fluorophore for quenchingthe fluorescence of the fluorophore.
 5. The SPN of claim 4, wherein theSPN for detecting peanut comprises a nucleotide sequence selected fromthe group consisting of sequences presented by SEQ ID NOs.:8-10.
 6. TheSPN of claim 4, wherein the SPN for detecting egg white comprises anucleotide sequence selected from the group consisting of sequencespresented by SEQ ID NOs.:11-14.
 7. The SPN of claim 4, wherein the SPNfor detecting wheat comprises a nucleotide sequence selected from thegroup consisting of sequences presented by SEQ ID NOs.:15-23.
 8. The SPNof claim 4, wherein the SPN for detecting milk comprises a nucleotidesequence selected from the group consisting of sequences presented bySEQ ID NOs.:24-26.
 9. A method for detecting one or more allergen(s) ina sample, comprising: (a) obtaining a test sample suspected ofcontaining said one or more allergen(s), (b) processing said test sampleand extracting proteins from the processed sample using an extractionbuffer, (c) mixing the extracted proteins with at least one signalingpolynucleotide (SPN) that comprises a nucleotide sequence selected fromthe group consisting of SEQ ID NOs. 8-26, (d) treating the mixture ofstep (c) with an excitation means, and (e) measuring the interactionbetween the signaling polynucleotide and the allergen(s) which indicatesthe absence or presence of the allergen(s) in the test sample, whereinthe SPN further comprises a fluorophore and a quencher which are insufficient proximity for the quencher to quench the fluorescence of thefluorophore.
 10. The method of claim 9, wherein the signalingpolynucleotide (SPN) comprises a nucleotide sequence or a portionthereof which specifically binds to the one or more allergen(s).
 11. Themethod of claim 10, wherein the binding of the SPN to the allergenseparates the quencher from the fluorophore, thereby allowing detectionof the fluorescence of the fluorophore.
 12. The method of claim 11,wherein the binding of the SPN to the allergen causes a change in thesecondary structure of the SPN.
 13. The method of claim 9, wherein thefluorophore and the quencher are linked to opposite ends of the sequenceof the signaling polynucleotide(SPN) and wherein 5 to 20 nucleobaseresidues at the 5′-end of the sequence are at least 80% complementary to5 to 20 nucleobase residues at the 3′-end of the sequence capable offorming a hairpin structure, thereby bringing the quencher in sufficientproximity to the fluorophore for quenching the fluorescence of thefluorophore.
 14. The method of claim 9 , wherein the SPN furthercomprises a linker sequence 5 to 20 nucleobases in length annealed tothe 5′-end of the sequence of the signaling polynucleotide(SPN) andhaving at least 80% complementarity with the 5′-end of the sequence ofthe aptamer, wherein the aptamer sequence comprises a fluorophore andthe linker sequence comprises a quencher, or wherein the aptamersequence comprises a quencher and the linker sequence comprises afluorophore.
 15. The method of claim 9, wherein the extraction bufferused in step (b) is a universal extraction buffer.
 16. The method ofclaim 9, wherein a total protein is measured for the processed sample.17. The method of claim 16, wherein the total protein is measured usingPyrogallol Red-molybdate (PRM) agent.
 18. The method of claim 17,wherein the total protein is determined as absorbance at 600 nm.
 19. Themethod of claim 9, wherein the excitation means is a light-emittingdiode (LED) light.
 20. The method of claim 9, wherein the test sample isa food sample.